Immobilized endoglycosidase fusion protein and application thereof

Abstract

The invention relates to a method for preparing an antibody-coupled drug, the antibody-coupled drug is subjected to fixed-point coupling based on an antibody Fc region N-glycosylation site, and the method comprises the following steps: (1) providing a donor containing oxazoline oligosaccharide, an antibody containing a GlcNAc motif and immobilized endoglycosidase with glucoside transfer activity; (2) covalently linking an activated donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif under the catalytic action of glycosidase; and one-step coupling is realized. The invention also relates to an endonuclease fusion protein which comprises the endonuclease, a Halo tag and / or a His tag which are covalently linked. The invention also relates to an immobilized glycosidase fusion protein obtained by immobilizing the glycosidase fusion protein on a support, a preloaded column filled with the immobilized fusion protein, and a purification method of the immobilized glycosidase fusion protein for an antibody conjugate.

Description

Immobilized endoglycosidase fusion protein and its application Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for preparing a drug conjugate or antibody-drug conjugate using an immobilized endoglycosidase fusion protein having glycoside transfer activity, wherein the antibody-drug conjugate is site-coupled in a single step based on an N-glycosylation site in the Fc region of an antibody. The present invention also relates to an endoglycosidase fusion protein comprising a covalently linked endoglycosidase, a Halo tag, and / or a His tag, and an immobilized endoglycosidase fusion protein obtained by immobilizing the endoglycosidase fusion protein on a support. Background Art

[0002] The demand for high-quality conjugates, particularly for bioconjugates used in bioscience research, diagnostics, or therapeutics, is rapidly increasing. However, the stable and high-quality production of bioconjugates remains far from satisfactory, partly due to the complexity of biomolecules and the difficulty of scale-up, which makes it difficult to achieve high-quality bioconjugate production standards.

[0003] Antibody-drug conjugates (ADCs) are a new type of targeted drug that falls under the category of bioconjugates. They organically connect highly active small molecule drugs to monoclonal antibodies through chemical linkage. They have both the high activity of small molecule drugs and the targeting of antibody drugs. They can better overcome the significant toxic side effects of small molecule toxins on the human body, while also overcoming the limited efficacy of antibody therapy for solid tumors, making them a class of anti-tumor drugs with great development prospects.

[0004] With the development and gradual maturity of antibody-drug conjugates, antibody conjugation technology has undergone several iterative upgrades, and has roughly experienced three generations of technology development, including random conjugation, site-specific conjugation based on antibody genetic engineering, and site-specific conjugation technology that does not rely on antibody genetic engineering (Walsh SJ, Bargh JD, Dannheim FM, etc., Site-selective modification strategies in antibody-drug conjugates, Chem. Soc. Rev. 2021, 50, 1305-1353). The first generation of conjugation technology mainly uses random conjugation based on lysine or cysteine ​​residues, and generally obtains a highly heterogeneous mixture with random conjugation sites and uneven drug-antibody ratio (DAR). There are problems in process stability, quality control, drug stability, metabolic consistency, safety, etc. The second generation of conjugation technology usually requires the introduction of specific amino acids into the antibody through engineered mutations or the insertion of polypeptide fragments of specific sequences. On this basis, the site-specific conjugation of the antibody is achieved through site-specific chemical conjugation or enzymatic conjugation. The third generation of coupling technology does not require the engineering modification of antibodies. It focuses on selecting specific amino acid sites and using specific conditions or proximity effects for site-specific coupling, including the modification of interchain disulfide bonds, chemical selectivity modification, modification based on proximity effects, and glycosylation. In comprehensive comparison, glycosylation has become a research hotspot in antibody coupling technology because it does not require antibody engineering modification, does not involve changes in the core skeleton structure of the antibody, does not affect the binding ability of the antigen-binding region of the antibody, does not require fine-tuning of chemical reaction conditions, and has good coupling repeatability and uniformity. There is a highly conserved glycosylation (N-297Glycan) at asparagine 297 in the Fc region of the antibody. The site-specific connection of different molecules on the antibody can be achieved through the remodeling of the sugar chain at this site (Wang LX, Tong X, Li C. Glycoengineering of Antibodies for Modulating Functions, Annu Rev Biochem, 2019, 88, 433-459). The site-specific coupling technologies based on antibody Fc glycosylation are mainly:

[0005] 1) Chemical modification based on glycosyl groups uses sodium periodate to oxidize core fucose (Zuberbuhler K, Casi G, Bernardes GJ, et al., Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in IgG format, Chem Commun, 2012, 48, 7100-7102) or the vicinal diol of the glycosyl-terminal sialic acid (Zhou Q, Stefano JE, Manning C, et al., Site-specific antibody–drug conjugation through glycoengineering, Bioconjugate Chem, 2014, 25, 510-520) to generate the corresponding aldehyde. The aldehyde carbonyl group can be used to conjugate with small molecule toxin fragments to prepare ADCs. However, due to the diversity of glycosyl groups at N-297, not all monoclonal antibodies contain core fucose or sialic acid reaction sites, resulting in certain substrate limitations.

[0006] 2) Enzyme-catalyzed modification, using the deglycosylation-transglycosylation relay catalysis of tool enzymes such as endoglycosidases and glycoside transferases to achieve sugar chain remodeling and introduce bioorthogonal reactive groups, and then through subsequent chemical reactions, achieve site-specific conjugation preparation of ADCs (Wang LX, Tong X, Li C, Glycoengineering of antibodies for modulating functions, Annu Rev Biochem, 2019, 88, 433-459; and Zeng Y, Tang F, Shi W et al., Recent advances in synthetic glycoengineering for biological applications, Current Opinion in Biotechnol, 2022, 74, 247-255).

[0007] In 2012, Wang Laixi et al. reported a site-specific conjugation technology for antibody glycan remodeling based on the relay catalysis of the endoglycosidase Endo S and its mutants, synthesizing a glycan-remodeled antibody with a single glycoform structure and containing azide modifications (Huang W, Giddens J, Fan SQ et al., Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions, J. Am. Chem. Soc., 2012, 134, 12308). Based on this work, Wang Laixi, Huang Wei et al. developed a large class of ADC site-specific conjugation synthesis technologies based on a three-step deglycosylation-transglycosylation-click chemistry reaction catalyzed by tool enzymes such as Endo S or Endo S2 and their mutants (Zeng Y, Tang F, Shi W et al., Recent advances in synthetic glycoengineering for biological applications, Current Opinion in Biotechnol, 2022, 1074, 247-255). In 2021, Wang Laixi et al. reported a one-pot Endo S2-catalyzed site-specific conjugation of antibody sugar chains, introducing bioorthogonal azide functional groups into the antibody, and then obtaining ADC molecules through a one-step click chemistry reaction (Zhang X, Ou C, Liu H et al., General and robust chemoenzymatic method for glycan-mediated site-specific labeling and conjugation of antibodies: facile synthesis of homogeneous antibody-drug conjugates, ACS Chem.Biol., 2021, 16, 11, 2502-2514).

[0008] Another commonly used tool enzyme in the site-specific coupling technology of sugar chain remodeling is β-1,4-galactosyltransferase (β-1,4-Gal-T1) and its mutant (β-1,4-Gal-T1Y289L). This enzyme uses uridine diphosphate galactose (Gal-UDP) as a donor to transfer galactose Gal to the non-reducing end of acetylglucosamine (Glc-NAc) of glycoproteins. Based on this strategy, in 2009, Qasba et al. (Boeggeman E, Ramakrishnan B, Pasek M et al., Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection, Bioconjugate Chem., 2009, 20, 6, 1228-1236) first reported the antibody site-specific conjugation technology using β-1,4-Gal-T1 and β-1,4-Gal-T1Y289L as tool enzymes and C2-keto-Gal-UDP or N-azidoacetylgalactosamine-UDP (GalNAz-UDP) as donors to prepare antibodies modified with ketocarbonyl or azido groups. Subsequently, in 2014, they reported the first ADC molecule synthesis method based on the three-step deglycosylation-transglycosylation-bioorthogonal reaction catalyzed by β-1,4-Gal-T1 and its mutants (Zhu Z, Ramakrishnan B, Li J et al., Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar, mAbs, 2014, 6, 1190-1200).Based on the above work, Synaffix developed a three-step ADC synthesis method of deglycosylation-transglycosylation-bioorthogonal reaction by combining the endoglycosidase Endo S and the tool enzyme β-1,4-Gal-T1Y289L for relay catalysis (Van Geel R, Wijdeven MA, Heesbeen R et al., Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates, Bioconjugate Chem., 2015, 26, 2233-2242). In addition, a four-step synthesis method for ADCs, which involves deglycosylation-transglycosylation-transglycosylation-bioorthogonal reaction, has been reported in the literature (Li X, Fang T, Boons GJ, Preparation of well-defined antibody–drug conjugates through glycan remodeling and strain-promoted azide–alkyne cycloadditions, Angew. Chem. Int. Ed., 2014, 53, 7179-7182). This method uses three tool enzymes, namely β-1,4-Gal-T1, β-1,4-Gal-T1Y289L and sialyltransferase.

[0009] The above-mentioned sugar chain remodeling coupling technologies all utilize multi-step reactions catalyzed by liquid-phase enzymes, which have certain limitations. For example, liquid-phase enzyme catalysis requires a large proportion of endoglycosidase catalytic equivalents, has high requirements for enzyme purity (complex process and high preparation cost), and is prone to introduce a large amount of enzyme-related impurities (such as host proteins and nucleic acids) into the reaction system, which poses a great challenge to the removal of impurities in the subsequent downstream of the coupled drug.

[0010] Currently, there are only a few publicly reported examples of sugar chain coupling reaction modes based on immobilized enzyme catalysis. For example, in 2018, Wang Laixi et al. reported a tandem column coupling based on covalently immobilized Endo S2 enzyme and covalently immobilized Endo S2D184M enzyme, which achieved continuous flow relay catalysis of deglycosylation-transglycosylation to obtain sugar-modified sugar chain homogeneous antibodies (Li T, Li C, Quan DN et al., Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies, Carbohydr. Res. 2018, 458-459, 77-84). In 2021, Wu Zongyi et al. reported the relay catalysis of liquid-phase suspended immobilized enzymes Endo S2 and Endo S2T138Q to obtain glycosylated sugar chain homogeneous antibodies (Chuang H, Huang C, Hung T, et al., Development of biotinylated and magnetic bead-immobilized enzymes for efficient glyco-engineering and isolation of antibodies, Bioorg. Chem. 2021, 112, 104863). There is currently no report on the one-step preparation of ADC drugs based on immobilized enzyme-catalyzed sugar chain remodeling. In short, the current existing sugar chain remodeling reaction avoids the problem of other site-directed coupling technologies requiring engineering modifications to antibodies, but there are still very large limitations, such as lengthy coupling steps and cumbersome operations. At least two enzymatic reactions plus one chemical reaction are required, that is, at least three steps of reaction and three complete purifications are required to obtain ADC. On the other hand, the vast majority of existing sugar chain remodeling is based on liquid-phase enzyme-catalyzed reactions, which have problems such as difficulty in scale-up, difficulty in separating and removing glycosidases, and great challenges in industrialization. According to literature reports, even trace amounts of residual tool enzymes in ADC products may cause ADC products to deglycosylate and release toxin molecules (Li T, Li C, Quan DN, et al., Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies, Carbohydrate Research 2018, 458-459, 77-84), which may cause serious toxic reactions, bring major challenges to drug development and production, and also bring major risks to ADC drug safety. The present invention aims to solve these problems.

[0011] Summary of the Invention

[0012] Antibody-drug conjugates can be conjugated using site-specific conjugation technology based on sugar chain remodeling. Asparagine at position 297 in the Fc region of antibodies is highly conserved and glycosylated. Sugar chain remodeling at this site allows for site-specific conjugation of different molecules to antibodies. Proteins containing the Fc region (such as Fc fusion proteins) can also be site-specifically conjugated to different molecules based on asparagine at position 297 in the Fc region of antibodies. The location of asparagine at position 297 in proteins containing the Fc region is based on the amino acid sequence of the antibody.

[0013] This application uses an immobilized endoglycosidase with glycosyltransferase activity to reshape the Fc-terminal sugar chain of an antibody, and then efficiently and specifically couples the linker-load to an Fc-containing protein or antibody through a suspension liquid phase catalysis or column continuous flow catalysis process, thereby obtaining an antibody-drug conjugate with good uniformity in one step.

[0014] In one aspect, the present invention provides a method for preparing a conjugated drug, wherein the conjugated drug is site-specifically conjugated to an N-glycosylation site in the Fc region, the method comprising the following steps:

[0015] (1) providing a donor containing an oxazoline oligosaccharide, a protein containing an Fc region, and an immobilized endoglycosidase having glycoside transfer activity, wherein the Fc region contains a GlcNAc motif;

[0016] (2) covalently linking the donor containing oxazoline oligosaccharide to the protein containing Fc motif through the catalytic action of the endoglycosidase.

[0017] In some embodiments, the protein containing the Fc region is an antibody or an Fc fusion protein. In one aspect, the present invention provides a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site of the antibody Fc region, the method comprising the following steps: (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase with glycoside transfer activity; (2) covalently linking the activated donor containing an oxazoline oligosaccharide to the antibody containing the GlcNAc motif through the catalytic action of the endoglycosidase.

[0018] In some embodiments, the donor containing oxazoline oligosaccharides further contains a load. In some embodiments, the load is selected from the group consisting of small molecule compounds, agonists, nucleic acids, nucleic acid analogs, fluorescent molecules, radionuclides, and immunomodulatory proteins (such as interleukins). In some embodiments, the load is selected from a group consisting of small molecule compounds (e.g., small molecule drugs of various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapy drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide, and bendamustine; small molecule targeted drugs, such as imatinib mesylate, gefitinib, and anlotinib; immune agonists, such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes, and radioactive labels, etc.), short peptides, polypeptides, peptidomimetics, and proteins.

[0019] In some embodiments, the oxazoline oligosaccharide is selected from one or more of the following groups: disaccharide oxazoline, trisaccharide oxazoline, tetrasaccharide oxazoline, pentasaccharide oxazoline, hexasaccharide oxazoline, heptasaccharide oxazoline, octasaccharide oxazoline, nonasaccharide oxazoline, decaasaccharide oxazoline, and undecaasaccharide oxazoline. In some embodiments, the oxazoline oligosaccharide has the following structure:

[0020] The first hexose group or its derivative-(the second hexose group or its derivative) f -β-D-pyranose glucoxazoline,

[0021] Wherein f is 0, 1, 2, 3, 4, 5 or 6; the structure of β-D-pyranose glucoxazoline is:

[0022] In some embodiments, the first hexosyl group or its derivative is selected from the group consisting of glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or their derivatives; and / or its 6-carbon is in the form of -C(O)-; and / or

[0023] The second hexose group or its derivative is independently selected from the group consisting of glucosyl, mannosyl, galactosyl, fructosyl or their derivatives at each occurrence; and / or the monosaccharide moieties in the oligosaccharide structure are linked by β-(1→4) glycosidic bonds; and / or

[0024] The derivatives (ie, the first hexagonal glycosyl derivative and the second hexagonal glycosyl derivative) are independently selected from derivatives in which the hydroxyl group of uronic acid or monosaccharide is replaced by an acylamino group.

[0025] In some embodiments, the oxazoline oligosaccharide has the following structure:

[0026] The first hexose group or its derivative - β-D-pyranose glucoxazoline, wherein the first hexose group or its derivative is mannitol (mannosyl) or its derivative.

[0027] In some embodiments, the oxazoline oligosaccharide has the following structure:

[0028] The first hexose group or its derivative is β-D-pyranose glucoxazoline, wherein the first hexose group or its derivative is galactose group or its derivative.

[0029] In some embodiments, the oxazoline oligosaccharide has the following structure:

[0030] The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is a glucose group or a derivative thereof; or

[0031] The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is a fructose group or a derivative thereof; or

[0032] The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is a gulose group or a derivative thereof; or

[0033] The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is an idose group or a derivative thereof.

[0034] In some embodiments, the structure of the oxazoline oligosaccharide is:

[0035] In some embodiments, the structure of the oxazoline oligosaccharide is:

[0036] In one aspect, the present invention provides a method for preparing a drug conjugate, wherein the antibody drug conjugate is site-specifically conjugated based on the N-glycosylation site of the Fc region, the method comprising the following steps:

[0037] (1) providing a donor containing an oxazoline oligosaccharide, a protein containing an Fc region, and an immobilized endoglycosidase having glycoside transfer activity, wherein the Fc region contains a GlcNAc motif;

[0038] The donor containing oxazoline oligosaccharide is a linker-load compound of formula (I):

[0039] (2) covalently linking the donor containing oxazoline oligosaccharides to the protein containing the Fc region through the catalytic action of the endoglycosidase;

[0040] Wherein, the drug-drug conjugate structure is shown in formula (II):

[0041] Wherein, P is the load;

[0042] DC(O)-L- is a linker;

[0043] The carbon 6 of the first hexose moiety or its derivative is in the form of -C(O)-, and f is 0, 1, 2, 3, 4, 5 or 6;

[0044] L is a linker, and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1;

[0045] R is hydrogen or α-L-fucosyl;

[0046] q is 1 or 2;

[0047] Protein is a protein containing an Fc region.

[0048] In some embodiments, -L-(P)t is represented by the following formula I-1 or formula I-2.

[0049] In some embodiments, the first hexosyl group or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or their derivatives; and / or

[0050] The second hexosyl group or its derivative is independently selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives at each occurrence; and / or

[0051] The monosaccharide moieties in the oligosaccharide structure are linked by β-(1→4) glycosidic bonds; and / or

[0052] The first hexose derivative and the second hexose derivative are independently selected from uronic acid or a derivative in which the hydroxyl group of a monosaccharide is replaced by an acylamino group. In some embodiments, the protein containing an Fc region is an antibody or an Fc fusion protein.

[0053] In one aspect, the present invention provides a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated to an N-glycosylation site in the Fc region of an antibody, the method comprising the following steps:

[0054] (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycoside transfer activity;

[0055] The donor containing oxazoline oligosaccharide is a linker-load compound of formula (I):

[0056] (2) covalently linking the donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif through the catalytic action of the endoglycosidase;

[0057] Wherein, the structure of the antibody-drug conjugate is as shown in formula (II-1), (II-2), (II-3), (II-4) or (II-5):

[0058] Wherein, P is the load;

[0059] DC(O)-L- is a linker;

[0060] DC(O)- is a disaccharide structure;

[0061] L is a linker, and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1;

[0062] R is hydrogen or α-L-fucosyl;

[0063] q is 1 or 2;

[0064] Ab is an antibody or an antigen-binding fragment thereof.

[0065] In one aspect, the present invention provides a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site of the antibody Fc region, and the method comprises the following steps:

[0066] (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycoside transfer activity;

[0067] The donor containing oxazoline oligosaccharide is a linker-load compound of formula (I):

[0068] (2) covalently linking the donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif through the catalytic action of the endoglycosidase;

[0069] The structure of the antibody-drug conjugate is shown in formula (II):

[0070] Wherein, P is the load;

[0071] DC(O)-L- is a linker;

[0072] DC(O)- is a disaccharide structure

[0073] L is a linker, and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1;

[0074] R is hydrogen or α-L-fucosyl;

[0075] q is 1 or 2;

[0076] Ab is an antibody or an antigen-binding fragment thereof.

[0077] In some embodiments, -L-(P) in formula (I) t -L 2 -L 1 -BP, in which case formula (I) is:

[0078] in

[0079] B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-;

[0080] L 1 independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;

[0081] L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination:

[0082] 1)-NH-C 2-20Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent;

[0083] 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or an integer from 10 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure;

[0084] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group;

[0085] P is with B part, or L 1 Part, or L 2In one aspect, the present invention provides a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site of the antibody Fc region, and the method comprises the following steps:

[0086] Provided are a linker-load compound of formula (I), an antibody, and an immobilized endoglycosidase having transglycosidic activity:

[0087] forming an antibody-drug conjugate through the catalytic action of the endoglycosidase;

[0088] Wherein, P is the load;

[0089] DC(O)-L- is a linker;

[0090] DC(O)- is a disaccharide oxazoline with the following structure:

[0091] A first hexose group or a derivative thereof - β-D-pyranose glucoxazoline, wherein the first hexose group or a derivative thereof is a galactosyl group or a derivative thereof;

[0092] L is a linker, and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1;

[0093] R is hydrogen or α-L-fucosyl;

[0094] q is 1 or 2;

[0095] Ab is an antibody or an antigen-binding fragment thereof.

[0096] In some embodiments, -L-(P) in formula (I) t -L 2 -L 1 -BP, in which case formula (I) is:

[0097] in

[0098] B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-;

[0099] L 1 independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;

[0100] L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination:

[0101] 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent;

[0102] 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or an integer from 10 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure;

[0103] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group;

[0104] P is with B part, or L 1 Part, or L 2 Partially connected load.

[0105] In some embodiments, wherein -L-(P)t is

[0106] That is, formula (I) is:

[0107] in,

[0108] Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -CO-(PEG) on the side chain j -R 11 a substituted natural amino acid or an oligomeric natural amino acid having a degree of polymerization of 2-10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10); R 11 It is C 1-10 alkyl;

[0109] d is 0, 1, 2, 3, 4, 5, or 6;

[0110] -(PEG) i -and-(PEG) j - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 Alkylene; each i is independently an integer from 1 to 100, and each j is independently an integer from 1 to 100;

[0111] M is hydrogen or LKa-L 2 ―L 1 ―B―P;

[0112] Q is NH2 or L 2 ―L 1 ―B―P;

[0113] Provided that the following is not included: M is hydrogen and at the same time Q is NH2;

[0114] Each LKa is independently selected

[0115] opSu is or a mixture thereof; wherein * represents and L 2 The connecting part;

[0116] B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-;

[0117] L 1 independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;

[0118] L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination:

[0119] 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent;

[0120] 2) Amino acid residue sequence, i.e. -*(AA) n**-, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or an integer from 10 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure;

[0121] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group;

[0122] P is with B part, or L 1 Part, or L 2 Partially connected load.

[0123] In some embodiments, L 2 is the amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * terminus forms an amide bond with the carbonyl group in the disaccharide structure. In some embodiments, AA is independently any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His, or any combination thereof at each occurrence.

[0124] In some embodiments, n is an integer from 1 to 100. In some embodiments, n is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 20, 22, 25, 28, 32, 34, 40, 50, 52, 60, 70, 86, 90, 100, or an interval (or endpoint) between any two values. In some embodiments, n is about 1 to 50. In some embodiments, n is about 1 to 30. In some embodiments, n is about 1 to 20. In some embodiments, n is about 1 to 10.

[0125] In some embodiments, L 1 is a cleavable sequence comprising an amino acid sequence that can be cleaved by an enzyme, wherein the amino acid sequence that can be cleaved by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In some embodiments, L 1 is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably, -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof. In some embodiments, L 1 For -Val-Cit-.

[0126] In some embodiments, wherein B is selected from: (-PABC-), -NH-CH2-U-, or -NH-CH2-U-(CH2) g -(CO)-; wherein g is 1, 2, 3, 4, 5 or 6; U is absent or is CH2, O, S or NH, preferably O or S.

[0127] In some embodiments, -L 1 -B- represents -Val-Cit-PABC-. ​​In some embodiments, -L 2 -L 1 -B- denotes -Gly-Gly-Gly-Val-Cit-PABC-.

[0128] In some embodiments, the payload P is selected from the group consisting of small molecule compounds (e.g., small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapy drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine, etc.; small molecule targeted drugs, such as imatinib mesylate, gefitinib and anlotinib, etc.; immune agonists, such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radioactive labels, etc.), short peptides, polypeptides, peptidomimetics and proteins. In some embodiments, the payload P is a cytotoxin or a fragment thereof, with optional derivatization to be attached to the L portion in the compound of formula (I).

[0129] In some embodiments, the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhy-drovinblastine, dolastatin 10, and vinblastine. 10) and its analogs, halichondrin B, eribulin, indole-3-oxyacetamides, podophyllotoxins, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide, camptothecins and their derivatives, mitoxantrone, mitoguanidine, nitrogen mustards, nitrosoureas, aziridines, benzodopa, carboquinone, metodepa, uredepa, dynemicin, esperamicin (esperamicin), neocarzinostatin, aclarubicin, dactinomycin, anthramycin, bleomycin, actinomycin C, caramidocin, carminomycin, carminomycin, dactinomycin D, daunorubicin, detoxib, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycins, noramycin, olivomycin, peplomycin, porfiromycin, puromycin, ferroxorubicin, rhodorubicin, streptozocin, streptozocin, zorubicin, trichothecenes, T-2 toxin, verracurin A, baculosporin A, anguidine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, dimethylfolate, methotrexate, pteropterin, trimetrexate, edatrexate, fludarabine, 6-mercaptopurine, thiamidine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, dooxyfluridine, floxuridine, captestosterone, drostanolone propionate, cyclothiosteroid, methotrexate, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprolide acetate, protein kinase inhibitors and proteasome inhibitors; and / or

[0130] The cytotoxin is selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine, and exatecan, and derivatives thereof; and / or

[0131] The cytotoxin is selected from auristatin, in particular MMAE, MMAF or MMAD; and / or

[0132] The cytotoxin is selected from exatecan and its derivatives, such as DX8951f; and / or

[0133] The cytotoxin is selected from the group consisting of DXd-(1) and DXd-(2); preferably DXd-(1).

[0134] In some embodiments, the linker-load compound is represented by formula (I-2):

[0135] In some embodiments, the antibody is selected from the group consisting of anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD30 / TNFRSF8 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD44v6 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD117 / KITk antibody, anti-CD123 antibody, anti-CD138 antibody, anti-CD142 antibody, anti-CD174 antibody, anti-CD227 / MUC1 antibody, anti-CD352 antibody, anti-CLDN18.2 antibody, anti-DLL3 antibody, anti-ErbB2 / HER2 antibody, anti-CN33 antibody, anti-GPNMB antibody, anti-ENPP3 antibody. Antibodies, anti-Nectin-4 antibody, anti-EGFRvⅢ antibody, anti-SLC44A4 / AGS-5 antibody, anti-CEACAM5 antibody, anti-PSMA antibody, anti-TIM1 antibody, anti-LY6E antibody, anti-LIV1 antibody, anti-Nectin4 antibody, anti-SLITRK6 antibody, anti-HGFR / cMet antibody, anti-SLAMF7 / CS1 antibody, anti-EGFR antibody, anti-BCMA antibody, anti-AXL antibody, anti-NaPi2B antibody, anti-GCC antibody, anti-STEAP1 antibody, anti-MUC16 antibody, anti-mesothelin antibody, anti-ETBR antibody, anti-EphA2 antibody, anti-5T4 antibody, anti-FOLR1 antibody, anti-LAMP1 antibody, anti-Cadherin 6 antibodies, anti-FGFR2 antibodies, anti-FGFR3 antibodies, anti-CA6 antibodies, anti-CanAg antibodies, anti-integrin αV antibodies, anti-TDGF1 antibodies, anti-ephrin (Ephrin) A4 antibodies, anti-TROP2 antibodies, anti-PTK7 antibodies, anti-NOTCH3 antibodies, anti-C4.4A antibodies, anti-FLT3 antibodies, anti-B7H3 / 4 antibodies, anti-TF (Tissue Factor) antibodies, anti-ROR1 / 2 / antibodies; preferably anti-CD19 antibodies, anti-ErbB2 / HER2 antibodies, anti-CLDN18.2 antibodies, anti-Nectin-4 antibodies, anti-FGFR3 antibodies, anti-Trop2 antibodies.

[0136] In some embodiments, the endoglycosidase having transglycosidic activity is N-acetylglucosamine endohydrolase. In some embodiments, the N-acetylglucosamine endohydrolase includes at least one selected from Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd) and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); or the N-acetylglucosamine endohydrolase includes at least one selected from Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S and Endo S2.

[0137] In some embodiments, the glycosidase having glycoside transfer activity is covalently linked to a Halo tag, and the glycosidase fusion protein is fixed to a support containing a haloalkyl linker via the Halo tag; the Halo tag is a dehalogenase or a variant thereof or a truncated functionally active portion thereof. In some embodiments, one end of the glycosidase is linked to a Halo tag, and the other end of the glycosidase is linked to a His tag (a His tag is a histidine polypeptide, such as His4, His5, His6, His8, His 10 , His 12 , or His 14 In some embodiments, the amino terminus of the endoglycosidase is connected to a Halo tag, and the carboxyl terminus of the endoglycosidase is connected to a His tag, i.e., Halo-endoglycosidase-His (His here refers to a His tag, which is His4, His5, His6, His8, His 10 , His 12 , or His 14 In some embodiments, the amino terminus of the endoglycosidase is connected to a Halo tag, and the carboxyl terminus of the endoglycosidase is connected to a His tag, and the endoglycosidase is Endo-S2, i.e., Halo-Endo S2-His.

[0138] In some embodiments, the support comprises a chloroalkyl linker such that the endoglycosidase fusion protein is immobilized on the support via a covalent interaction between the chloroalkyl linker and the Halo tag. In some embodiments, the chloroalkyl linker is generated from a chloroalkyl substrate having the structure of formula (III):

[0139] Wherein, u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19.

[0140] In some embodiments, the support has the structure of Formula (IV):

[0141] Wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19;

[0142] is a resin, bead, membrane, gel, matrix, film, plate, well, tube, slide, or surface. In some embodiments, In some embodiments, is agarose resin, silicone resin, polymethyl methacrylate resin or cellulose resin. In some embodiments, It is a highly cross-linked agarose resin or polymethyl methacrylate resin.

[0143] In one aspect, the present invention provides an endoglycosidase fusion protein comprising a covalently linked endoglycosidase and a Halo tag; the Halo tag is a dehalogenase or a variant thereof or a truncated functionally active portion thereof. In some embodiments, the endoglycosidase fusion protein comprises a covalently linked endoglycosidase and a Halo tag.

[0144] In some embodiments, the fusion protein is covalently linked to a Halo tag at one end of the endoglycosidase and a His tag at the other end of the endoglycosidase. In some embodiments, the amino terminus of the endoglycosidase is covalently linked to a Halo tag, and the carboxyl terminus of the endoglycosidase is covalently linked to a His tag.

[0145] In some embodiments, the endoglycosidase is selected from at least one of Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd) and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); or the endoglycosidase is selected from at least one of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S and Endo S2.

[0146] In one embodiment, the endoglycosidase is endo-β-N-acetylglucosaminidase. In one embodiment, the endoglycosidase is selected from the group consisting of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2.

[0147] In some embodiments, the His tag is a plurality of consecutive histidine residues. In some embodiments, the His tag is 3 histidines, 4 histidines, 5 histidines, 6 histidines, 7 histidines, 8 histidines, 9 histidines or 10 amino acids. In some embodiments, the His tag is His6. In one embodiment, the His tag is His8. In one embodiment, the His tag is His 10 .

[0148] In one embodiment, the endoglycosidase fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, or has at least 80% identity, at least 85% identity, at least 90% identity compared to SEQ ID NO: 1, or has one or more conservative amino acid substitutions compared to SEQ ID NO: 1.

[0149] In some embodiments, the endoglycosidase fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0150] In some embodiments, the endoglycosidase fusion protein comprises the amino acid sequence as shown in positions 1 to 1150 of SEQ ID NO: 1, or has at least 90% identity with the amino acids shown in positions 1 to 1150 of SEQ ID NO: 1, or has one or more conservative amino acid substitutions compared to the amino acids shown in positions 1 to 1150 of SEQ ID NO: 1. In some embodiments, the endoglycosidase fusion protein comprises or consists of the amino acid sequence as shown in positions 1 to 1150 of SEQ ID NO: 1.

[0151] In some embodiments, the endoglycosidase fusion protein has a PI of about 4 to 7. In some embodiments, the endoglycosidase fusion protein has a PI of about 4, 4.1, 4.3, 4.5, 4.7, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, or any two values ​​therebetween (including the endpoints).

[0152] In one aspect, the present invention provides a method for preparing the endoglycosidase fusion protein of the present invention, comprising: (a) providing a nucleic acid sequence of an endoglycosidase; (b) connecting a nucleic acid sequence of a Halo tag to one end of the nucleic acid sequence of the endoglycosidase;

[0153] The obtained nucleic acid sequence is cloned into a suitable vector, and the vector is transformed into a suitable host cell, and the endoglycosidase fusion protein of the present invention is expressed in the host cell.

[0154] In some embodiments, the method for preparing the glycosidase fusion protein of the present invention comprises: (a) providing a nucleic acid sequence of a glycosidase; (b) connecting a nucleic acid sequence of a Halo tag to one end of the nucleic acid sequence of the glycosidase; and (c) connecting a nucleic acid sequence of a His tag to the other end of the nucleic acid sequence of the glycosidase.

[0155] In some embodiments, the nucleic acid sequence of the Halo tag is linked to the amino terminus of the sequence of the endoglycosidase. In some embodiments, the nucleic acid sequence of the Halo tag is linked to the amino terminus of the nucleic acid sequence of the endoglycosidase, and the nucleic acid sequence of the His tag is linked to the carboxyl terminus of the nucleic acid sequence of the endoglycosidase.

[0156] In one aspect, the present invention provides an immobilized endoglycosidase fusion protein, which comprises the endoglycosidase fusion protein of the present invention immobilized on a support.

[0157] In some embodiments, the support comprises a haloalkyl linker, such that the endoglycosidase fusion protein is immobilized on the support via a covalent interaction between the haloalkyl linker and a Halo tag.

[0158] In some embodiments, the support comprises a chloroalkyl linker such that the endoglycosidase fusion protein is immobilized on the support via a covalent interaction between the chloroalkyl linker and the Halo tag. In some embodiments, the chloroalkyl linker is generated from a chloroalkyl substrate having the structure of formula (III):

[0159] Wherein, u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19.

[0160] In some embodiments, the support has the structure of Formula (IV):

[0161] Wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19;

[0162] is a resin, bead, membrane, gel, matrix, film, plate, well, tube, slide, or surface. In some embodiments, In some embodiments, is agarose resin, silicone resin, polymethyl methacrylate resin or cellulose resin. In some embodiments, It is a highly cross-linked agarose resin or polymethyl methacrylate resin.

[0163] On the other hand, the present invention also provides a prepacked column filled with immobilized endoglycosidase fusion protein.

[0164] On the other hand, the present invention also provides the use of the immobilized endoglycosidase fusion protein and / or the prepacked column in the preparation and / or purification of a conjugate drug or antibody conjugate. In some embodiments, the immobilized endoglycosidase fusion protein and / or the prepacked column are easy to scale up industrially in the preparation (e.g., purification) of the conjugate drug or antibody conjugate.

[0165] Advantages of the one-step coupling system catalyzed by the immobilized endoglycosidase of this application:

[0166] 1) Liquid-phase enzyme catalysis requires a large amount of endoglycosidase, which also introduces a large number of enzyme-related impurities (such as host proteins and nucleic acids) into the reaction system. This places high demands on enzyme purity (complex process and high preparation cost), and poses a great challenge to the removal of impurities in the subsequent drug conjugation downstream. Enzyme immobilization does not require high purity of the starting endoglycosidase (the enzyme preparation process is simple and low cost), and the impurities introduced into the reaction system are very few, greatly simplifying the impurity removal process downstream of the drug conjugation. In addition, enzyme immobilization facilitates separation and purification, making it easier to remove the tool enzyme from the product, reducing the possibility of product sugar chains and small molecules falling off due to tool enzyme residues, thereby reducing potential toxicity caused by product decomposition.

[0167] 2) Enzyme immobilization facilitates the reuse of enzymes;

[0168] 3) Continuous flow intelligent coupling technology greatly simplifies the reaction and purification process, significantly improves the efficiency of drug discovery, and ensures process robustness during drug production;

[0169] 4) One-step coupling of the antibody and the linker-load using an endoglycosidase immobilized on the column;

[0170] 5) The PI of the endoglycosidase fusion protein of the present invention is approximately 5.5, while the PI of the ADC conjugate is 8-9; the use of AEX and / or CEX facilitates the subsequent removal of the endoglycosidase fusion protein from the ADC product;

[0171] 6) The endoglycosidase fusion protein-coupling column catalysis of the present invention can achieve excellent coupling efficiency, is easy to linearly amplify, and residual enzyme is easily removed, thus being more suitable for the industrial production of ADC. BRIEF DESCRIPTION OF THE DRAWINGS

[0172] Figure 1 is an SDS-PAGE electrophoresis diagram showing the purified endoglycosidase fusion protein (Halo-Endo S2-His) of the present invention; in the figure, 1 represents a sample from a cell fluid, 2 represents a sample from a supernatant, 3 represents a sample from a flow-through (the components of the equilibration buffer used are: 50mM Tris, 150mM NaCl, 20mM imidazole, pH 7.4), 4 represents a sample from a washing solution (the components of the washing solution are: 50mM Tris, 150mM NaCl, 80mM imidazole, pH 7.4), 5 represents a sample from an eluate (the components of the eluate are: 50mM Tris, 150mM NaCl, 500mM imidazole, pH 7.4), 6 represents a sample from an eluate (the components of the eluate are: 50mM Tris, 150mM NaCl, 500mM imidazole, pH 7.4), and 7 represents a sample from magnetic beads; endoglycosidase fusion protein (Halo-Endo S2-His) S2-His) has a molecular weight of approximately 130 kDa;

[0173] FIG2 is an SDS-PAGE electrophoresis diagram showing the supernatant of the immobilized endoglycosidase fusion protein (Halo-Endo S2-His) of the present invention at different incubation times;

[0174] FIG3 is an SDS-PAGE electrophoresis diagram showing the use of the endoglycosidase fusion protein (Halo-Endo S2-His) of the present invention in a suspension coupling reaction;

[0175] FIG4 is a HIC-HPLC spectrum showing the product of the endoglycosidase fusion protein (Halo-Endo S2-His) of the present invention used in a suspension coupling reaction;

[0176] FIG5A is a graph showing the purification of ADC by Q Sepharose FF, and FIG5B is a graph showing the purification of ADC and endoglycosidase fusion protein by Q Sepharose FF;

[0177] FIG6A is a graph showing the purification of ADC after hanging column Capto S impact, and FIG6B is a graph showing the purification of ADC and endoglycosidase fusion protein after hanging column Capto S impact;

[0178] FIG7 shows the affinity detection of ADC-1 to ErbB2 / Her2 on the cell surface (EC50, nM);

[0179] Figure 8 shows the inhibitory effects of different drugs on the proliferation of tumor cells BT474 (IC50, nM);

[0180] Figure 9 shows the inhibitory effects of different drugs on the proliferation of tumor cells NCI-N87 (IC50, nM);

[0181] FIG10 shows the inhibitory effects of different drugs on the proliferation of tumor cells HepG2 (IC50, nM).

[0182] FIG11 shows the inhibitory effect of ADC-1 on the NCI-N87CDX mouse model.

[0183] Figure 12 shows the HIC-HPLC spectrum of ADC-2. Figure 13 shows the inhibitory effects of ADC-2 and different drugs on the proliferation of tumor cells SK-BR-3 (IC50, nM).

[0184] FIG14 shows the inhibitory effects of ADC-2 and different drugs on the proliferation of tumor cells HCC1954 (IC50, nM).

[0185] FIG15 shows the inhibitory effects (IC50, nM) of ADC-2 and different drugs on the proliferation of tumor cells MDA-MB-468.

[0186] FIG16 shows the deconvoluted high-resolution mass spectrum of ADC-3.

[0187] FIG17 shows the inhibitory effects of ADC-3 and different drugs on the proliferation of tumor cells BxPC-3 (IC50, nM).

[0188] FIG18 shows the inhibitory effects of ADC-3 and different drugs on the proliferation of FaDu tumor cells (IC50, nM).

[0189] FIG19 shows the inhibitory effects of ADC-3 and different drugs on the proliferation of tumor cells HepG2 (IC50, nM).

[0190] FIG20 shows the tumor inhibitory effect of ADC-3 on NCI-N87CDX mice.

[0191] FIG21 shows the effect of ADC-3 on body weight in NCI-N87CDX mice.

[0192] FIG22 is a diagram showing HIC-HPLC analysis of ADC-4.

[0193] FIG23 is a diagram showing HIC-HPLC analysis of ADC-5.

[0194] FIG24 shows the in vitro inhibitory activity of ADC-4 and ADC-5 against BxPC-3.

[0195] FIG25 shows the in vitro inhibitory activity of ADC-4 and ADC-5 against FaDu.

[0196] FIG26 shows the in vitro inhibitory activity of ADC-4 and ADC-5 against HepG2-3.

[0197] FIG27 shows the HIC-HPLC detection analysis of ADC-6.

[0198] FIG28 shows the in vitro inhibitory activity of ADC-6 against SK-BR-3.

[0199] FIG29 shows the in vitro inhibitory activity of ADC-6 against NCI-N87.

[0200] 1. Definition

[0201] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. In addition, terms and experimental methods related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology and immunology are terms and common methods widely used in the art. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient. All patents, published patent applications and publications cited herein are incorporated herein by reference. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0202] As used herein, the expression "at least one" or "one or more" or "a" or "a plurality" refers to one, two, three, four, five, six, seven, eight, nine, one hundred, two hundred, three hundred, four hundred, five hundred, six hundred, seven hundred, eight hundred, nine hundred or more, etc. As used herein, unless expressly stated otherwise, "a" and "an" should be understood to mean "at least one" or "at least one".

[0203] When a specific amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit or a preferred lower numerical limit, it should be understood as being equivalent to specifically disclosing any range by combining any numerical upper limit or preferred numerical value with any numerical lower limit or preferred numerical value, regardless of whether the range is explicitly listed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range, all integers and fractions (decimals) within the range, and the interval between any two numerical values. For example, the expression "u" is an integer from 1 to 20 should be understood as u being any integer from 1 to 20, for example, u can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Other similar expressions should also be understood in a similar manner.

[0204] The terms "about" and "approximately," when used in connection with a numerical variable, such as concentration, isoelectric point (pI), pH, temperature, or a specified range, generally mean that the value of the variable and all values ​​of the variable are within the range of experimental error (e.g., within a 95% confidence interval about the mean) or within ±10% of the specified value, or a wider range.

[0205] The term "optional" or "optionally" means that the subsequently described event may or may not occur, and that the description includes situations in which the event or circumstance occurs or does not occur.

[0206] The terms "comprising," "including," "containing," and "having" are open ended and do not exclude additional unrecited elements, steps, or ingredients. The term "consisting of excludes any element, step, or ingredient not specified. The term "consisting essentially of means that the scope is limited to the specified elements, steps, or ingredients, plus optional elements, steps, or ingredients that do not materially affect the key and novel characteristics of the claimed subject matter. It should be understood that the term "comprising" encompasses the terms "consisting essentially of" and "consisting of."

[0207] As used herein, the definition of "biomolecule" encompasses proteins, nucleic acids, lipids, carbohydrates, small nucleotides, amino acids, and their derivatives.

[0208] As used herein, "nucleic acid" or "polynucleotide" refers to a polymer of at least two nucleotides or nucleotide derivatives linked together by phosphodiester bonds, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0209] As used herein, " vector " is a kind of vehicle for transferring exogenous nucleic acid into host cell, and exogenous nucleic acid increases or expresses in host cell.As used herein, the definition of " vector " covers plasmid (such as linearized plasmid), viral vector, clay, phage vector, phagemid, artificial chromosome (such as, yeast artificial chromosome and mammalian artificial chromosome) etc..As used herein, vector can be expressed and / or replicated in host cell, refers to that vector can express RNA polynucleotide or polypeptide and / or produce multiple copies of vector in host cell.For " expressible " or " replicable ", vector can include nucleotide sequence or element that can be operably connected with promoter or replicon.As used herein, " operably connected " relevant to nucleotide sequence or element refers to that these nucleotide sequences are functionally related to each other.For example, promoter can be operably connected with the nucleotide sequence of coding polypeptide, and thus promoter regulates or mediates the transcription of nucleic acid.Those skilled in the art can select and use suitable vector according to specific purpose.

[0210] Fc fusion proteins are recombinant proteins produced by fusion of a biologically active protein or peptide with the hinge region or Fc fragment of IgG. These biologically active proteins or peptides include ligands, cytokines, receptors, antigens, and cyclic polypeptides that bind to cell surface antigens. Fc fusion proteins impart additional antibody-like properties, including extended plasma half-life and the unique functional effects of the Fc fragment.

[0211] The GlcNAc motif refers to a sugar chain with two conserved N-glycosylation sites at aspartic acid (Asn) 297 in the Fc region of an antibody. These sugar chains consist of N-acetylglucosyl-β-(1,4)-N-acetylglucosyl-glucosyl groups covalently linked to the Asn. Fc region glycosylation is a complex post-translational modification process that involves the formation of diverse sugar chains of varying lengths, compositions, and structures under the influence of various enzymes. These sugar chains have varying effects on protein bioactivity, conformation, stability, solubility, and pharmacokinetics. In some embodiments, Fc glycosylated carbohydrate molecules possess a complex biantennary core structure composed of two pentose sugars, mannose and N-acetylglucosamine. In addition to the core structure, different glycoforms contain varying numbers of additional sugars, such as fucose, mannose, N-acetylglucosamine, galactose, bisected N-acetylglucosamine, and sialic acid. Heterogeneity is often due to the galactosylation and sialylation of terminal sugars. Based on the number of terminal galactose residues, peptides are classified into three distinct subtypes (G0, G1, and G2). Each subtype contains different forms of core fucose and bisected N-acetylglucosamine, resulting in a total of 16 neutral complex structures. As used herein, "peptide," "polypeptide," or "protein" refers to two or more amino acids covalently linked. Unless otherwise specified, these terms are interchangeable.

[0212] "Amino acids" refer to organic compounds containing both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in the form of precursors. Individual amino acids are encoded by nucleic acids consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

[0213] As used herein, "sequence identity" has a meaning recognized in the art, and the percentage of sequence identity between two polypeptides can be calculated by aligning the two sequences using publicly available algorithms, such as BLAST and FASTA (see, e.g., Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994). Although there are many ways to measure the identity between two polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0214] As used herein, the term "variant" refers to a protein (or nucleic acid) having a substitution, deletion, or insertion of one or more residues (or nucleotides) compared to a reference protein (or nucleic acid). The reference protein can be a naturally occurring protein (i.e., a wild-type protein) that can be isolated from a natural resource, or an engineered protein. As used herein, the function or activity of a variant, such as an EndoS variant, an EndoS2 variant, or a Halo tag variant, is substantially similar, comparable, or higher than the function or activity of a reference EndoS, EndoS2, or Halo tag, respectively.

[0215] As used herein, the term "endoglycosidase" refers to an enzyme that catalyzes the hydrolysis of internal glycosidic bonds in oligosaccharide chains and polysaccharides, and can be used to cleave polysaccharides from glycoproteins. In some embodiments, the "endoglycosidase" of this application can be used to hydrolyze the β-(1→4) glycosidic bond between two N-acetylglucose residues at the N-glycosylation site in the Fc region of an antibody. "Glycoside transfer activity" refers to the catalytic attachment of activated sugars to various acceptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids, and small molecules. In this application, donors containing oxazoline oligosaccharides can be attached to the GlcNAc residues in the Fc region of an antibody.

[0216] As used herein, the term "Halo tag" refers to a haloalkane dehalogenase or a variant thereof that dehalogenates a haloalkane from a haloalkyl substrate (e.g., comprising a haloalkyl moiety -(CH2) 2-30-X, wherein X is a halogen such as F, Cl, Br, I, in particular Cl or Br) to remove the halogen and form a covalent bond with the remainder of the substrate. Mutated haloalkane dehalogenases have been described, for example, in WO2006 / 093529 and WO2008 / 054821, the relevant contents of which are incorporated herein by reference. Mutant haloalkane dehalogenases useful in the present invention may include, but are not limited to, mutants of Xanthobacter dehalogenases (e.g., Xanthobacter autotrophicus dehalogenase (DhIA)) or Rhodococcus dehalogenases (e.g., Rhodococcus rhodochrous dehalogenase (DhaA)), such as those comprising one or more substitutions at the catalytic triad residues, such as substitutions of His272 with Phe / Ala / Gly / Gln / Asn or substitutions of Asp106 with Cys or other substitutions, as described in WO2008 / 054821. The mutated haloalkane dehalogenase is capable of forming a covalent bond with a haloalkyl substrate.

[0217] "His tag" is a polypeptide composed of histidine, such as His-His(His2), His-His-His(His3), His-His-His-His(His4), His-His-His-H is-His(His5)、His-His-His-His-His-His(His6)、His-His-His-His-His-His-His-His-His(His 10 ).

[0218] As used herein, the term "fusion protein" refers to a protein product obtained by co-expression of two or more genes obtained by DNA recombination technology. For example, by deleting the stop codon of the gene encoding the first protein and then connecting a second protein gene with a stop codon, the co-expression of the two genes can be achieved. The endoglycosidase fusion protein may further comprise one or more additional elements, such as additional polypeptides or tags, such as Halo tags and / or His tags. In some embodiments, the endoglycosidase fusion protein substantially retains the desired properties.

[0219] As used herein, the term "resin" refers to an organic polymer that softens or melts when heated, tends to flow under external force when softened, and is solid, semi-solid, or liquid at room temperature. "Halogenated resin" refers to a new compound formed by replacing at least one functional group in a resin with a halogen group.

[0220] As used herein, the term "coupled" refers to the covalent linking of at least two moieties (eg, at least two molecules or at least two ends of the same molecule).

[0221] As used herein, a "conjugate" can be prepared by the covalent linkage of at least two moieties (eg, at least two molecules or at least two termini / side chains of the same molecule).

[0222] As used herein, the term "antibody-drug conjugate" refers to a conjugate comprising an antibody or antibody fragment covalently conjugated to a loading substance, which is a small molecule compound, an agonist, a nucleic acid, a nucleic acid analog, or a fluorescent molecule.

[0223] As used herein, the term "drug conjugate" refers to a conjugate comprising a protein containing an Fc region covalently coupled to a cargo, which is a small molecule compound, an agonist, a nucleic acid, a nucleic acid analog, or a fluorescent molecule.

[0224] The term "antibody" includes a wide variety of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that the classes of heavy chains include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4). The properties of this chain determine the "class" of the antibody, IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and the functional specificities conferred are also known. All immunoglobulin classes are within the scope of protection disclosed herein. In some embodiments, the immunoglobulin molecule is of the IgG class. IgG typically comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000. These four chains are connected in a "Y" configuration by disulfide bonds, with the light chains starting at the mouth of the "Y" and continuing through the variable region to surround the heavy chains.

[0225] Light chains can be classified as either kappa (κ) or lambda (λ). Each heavy chain can be bound to either a κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bound together, and the "tails" of the two heavy chains are bound together by covalent disulfide bonds or non-covalent bonds. Within the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the base of each chain. The variable region of an immunoglobulin κ light chain is Vκ; the variable region of an immunoglobulin λ light chain is Vλ.

[0226] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in accordance with the function. The variable regions of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of the constant regions increases as they become more distal to the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains comprise the carboxyl termini of the heavy and light chains, respectively.

[0227] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that form the antigen binding domain, assuming the antibody assumes its three-dimensional configuration in an aqueous environment, and that specifically bind to the antigen. The remaining amino acids in the antigen binding domain, referred to as the "framework" region, exhibit less intermolecular variability. The framework regions largely adopt a β-sheet conformation, with the CDRs forming loops connected thereto, or in some cases forming part of the β-sheet structure. Thus, the framework regions, by forming a scaffold, position the CDRs in the correct orientation through interchain non-covalent interactions. The antigen binding domain, with its specifically positioned CDRs, forms a surface complementary to an epitope on the antigen, which promotes non-covalent binding between the antibody and its antigenic epitope. For a given heavy or light chain variable region, one of ordinary skill in the art can identify the amino acids comprising the CDRs and framework regions by known methods (see Kabat, E. et al., US Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0228] The CDRs defined by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, the application of any definition to refer to the CDRs of an antibody or its variants is within the scope of the present invention. The exact residue numbering comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues the CDR comprises based on the variable region amino acid sequence of the antibody. Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence without relying on other experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., US Pat. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU numbering system.

[0229] The light chain constant region "comprises a portion of the amino acid sequence from an antibody light chain. Preferably, the light chain constant region (CL) comprises at least one of a constant kappa domain or a constant lambda domain. A "light chain-heavy chain pair" refers to a collection of light chains and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0230] The "Fc region" is the tail region of an antibody that interacts with cell surface receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments, derived from the second and third constant regions of the antibody's two heavy chains; in IgM and IgE antibody isotypes, the Fc region contains three heavy chain constant domains (CH2-4); the Fc region of IgG has highly conserved N-glycosylation sites. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity, and different glycoforms have different effects on the pharmaceutical properties of therapeutic antibodies.

[0231] Antibodies can be prepared using conventional recombinant DNA techniques. Cell lines producing antibodies can be selected, constructed, and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. In this regard, technical references suitable for use in the present invention described below include Current Protocols in Immunology, edited by Coligan et al., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DL Hacker, FMWurm, in Reference Module in Life Sciences, 2017; the entire contents of which, including supplementary content, are incorporated herein by reference.

[0232] In some embodiments, DNA encoding the antibody can be designed and synthesized according to conventional methods based on the antibody amino acid sequence described herein, inserted into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector comprises at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on either side of the inserted sequence. Efficient transcription can be achieved using the early and late promoters of SV40, and the early promoters of long terminal repeats from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus. Other cellular promoters, such as the actin promoter, can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.

[0233] In some embodiments, the inserted gene fragment needs to contain a selection marker. Common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and other selection markers to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking these genes. After culture in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein.

[0234] As used herein, the term "isoelectric point (pI)" is the pH (hydrogen concentration index) value of an aqueous solution of a molecule (e.g., a protein) when it has no net surface charge and is expressed in pH units. The pI of a protein can be measured experimentally using methods well known in the art, such as imaging capillary isoelectric focusing (iCIEF) and capillary isoelectric focusing (CIEF). Different biomolecules (proteins, nucleic acids, polysaccharides, etc.) with different pIs may have different charges at a given pH, thereby allowing their separation by methods such as ion exchange chromatography or isoelectric focusing.

[0235] As used herein, a molecule having a "basic pi" means that the molecule has a pi above 7.0. As used herein, a molecule having an "acidic pi" means that the molecule has a pi below 7.0.

[0236] As used herein, "ion exchange chromatography (IEX)" refers to a technique for separating biomolecules based on differences in their net surface charge and their affinity for an ion exchanger (also known as a medium, resin, or stationary phase). For example, in anion exchange chromatography, a protein with a pI lower than the buffer pH will have a negative net surface charge and bind to the positively charged anion exchanger; however, another protein with a pI higher than the buffer pH will have a positive net surface charge and will not bind to the positively charged anion exchanger, and therefore will pass through the medium along with the buffer.

[0237] As used herein, the term "support" refers to a water-insoluble material, such as a surface, gel, polymer, matrix, particle, resin, bead, or membrane, that can be separated from a reaction mixture in solid or semisolid form.

[0238] "Spacer" refers to a structure located between different structural modules that can spatially separate the structural modules. The definition of a spacer does not limit whether it has a certain function, nor does it limit whether it can be cut or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, wherein the non-amino acid structure can be, but is not limited to, an amino acid derivative or analog. "Spacer sequence" refers to an amino acid sequence that serves as a spacer, examples of which include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids, such as GA, or for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc. A self-immolative spacer (e.g., self-immolative spacer Sp1) is a covalent component that causes two chemical bonds to be cleaved sequentially after activation of a protective portion in the precursor: the protective portion (e.g., the cleavable sequence) is removed after activation, triggering a cascade of decomposition reactions, resulting in the release of smaller molecules in a sequential order. Examples of self-immolative spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0239] The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. Alkyl groups can have 1 to 20 carbon atoms and refer to "C1-C 20 "alkyl", such as C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, C3-C6 alkyl. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1 -dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or isomers thereof. A divalent free radical refers to a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons of the corresponding monovalent free radical. A divalent free radical has two attachment sites to the rest of the molecule. For example, "alkylene" or "alkylene group" refers to a saturated straight or branched divalent hydrocarbon group. Examples of "alkylene" include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexamethylene (-C6H 12-), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene or ethylpropylene, etc.

[0240] The term "cycloalkyl" refers to a cyclic saturated aliphatic group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. A cycloalkyl group can have 3 to 10 carbon atoms, i.e., "C3-C 10 "Cycloalkyl" refers to a divalent cycloalkyl radical, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. "Cycloalkylene" refers to a divalent cycloalkyl radical.

[0241] The term "heterocyclyl" refers to a cycloalkyl group in which one or more carbon atoms are replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, such as azepine, oxa-, or thiirane, azepine, oxa-, or thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, or tetrahydrothiopyranyl. "Heterocyclylene" refers to a divalent cycloalkyl group.

[0242] When "substituted" is mentioned herein, unless otherwise indicated, the relevant substituent is selected from alkyl, halogen, amino, monoalkylamino, dialkylamino, nitro, cyano, formyl, alkylcarbonyl, carboxyl, alkyloxycarbonyl, alkylcarbonyloxy, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, formylamino, alkylcarbonylamino, formyl(monoalkyl)amino or alkylcarbonyl(monoalkyl)amino.

[0243] As used herein, when a group is combined with another group, the connection between the groups can be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination can be connected to other parts of the molecule through any suitable atom in the structure, preferably through a specified chemical bond. For example, when two or more groups selected from -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 The heterocyclic group and the divalent group of -(CO)- are combined to form a combination, and two or more divalent groups can form a linear connection with each other, such as -CR 1 R 2 -C 1-10 Alkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene-C 1-10 Alkylene-(CO)-, -CR1 R 2 -CR 1 R 2’ -(CO)-、-CR 1 R 2 -CR 1 'R 2 '-CR 1” R 2” -(CO)-, etc. The resulting bivalent structure can be further linked to other parts of the molecule.

[0244] 2. Linker-Loader Compounds

[0245] In one aspect, the present invention provides a linker-load compound having formula (I):

[0246] in

[0247] P is the load;

[0248] DC(O)-L- is a linker;

[0249] DC(O)- is a disaccharide structure or;

[0250] L is a linker (for example, it can be disconnected from P chemically (for example, hydrolytically) or biologically (for example, enzymatically) to release P), and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0251] In some embodiments, -L-(P) t for

[0252] That is, formula (I) is:

[0253] in,

[0254] Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -CO-(PEG) on the side chain j- R 11 Substituted natural amino acids or oligomeric natural amino acids having a degree of polymerization of 2-10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0255] -(PEG)i -and-(PEG) j - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 alkylene;

[0256] M is hydrogen or LKa-L 2 ―L 1 ―B―P;

[0257] Q is NH2 or L 2 ―L 1 ―B―P;

[0258] Provided that the following is not included: M is hydrogen and Q is NH2;

[0259] Each LKa is independently selected

[0260] opSu is or a mixture thereof; wherein * represents and L 2 The connecting part;

[0261] Each L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination:

[0262] 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent;

[0263] 2) amino acid residue sequence, i.e., -*(AA)n**-, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure;

[0264] L 1 independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;

[0265] Each B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-;

[0266] P is with B part, or L 1 Part, or L 2 partially connected loads;

[0267] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group;

[0268] R 11 It is C 1-10 alkyl;

[0269] d is 0, 1, 2, 3, 4, 5, or 6;

[0270] Each i is independently an integer from 1 to 100; in some embodiments, i is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 20, 30, 34, 40, 50, 60, 70, 90, 100, or an interval between any two values ​​(including the endpoints);

[0271] Each j is independently an integer from 1 to 100; in some embodiments, j is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 20, 30, 34, 40, 50, 60, 70, 90, 100, or an interval between any two values ​​(including the endpoints); in some embodiments, j is about 1 to 20; in some embodiments, j is about 8 to 12; in some embodiments, j is about 8, 9, 12 or 13.

[0272] In one embodiment, B, L 1 and L 2 At least one of them is not "not present".

[0273] In one embodiment, L 2 Selected from: -NH-(CH2) a -(CH2)2(CO)-, a is an integer of 0, 1, 2, 3, 4 or 5; b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, L 2 The carbonyl group and L in the structure 1 In one embodiment, a is 0, 1, 2 or 3, preferably 3.

[0274] In one embodiment, L 2 is the amino acid residue sequence, i.e. -*(AA) n **-, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p-, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure. In one embodiment, AA is independently any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His or any combination thereof; in one embodiment, n is an integer from 2 to 100, preferably an integer from 2 to 50, preferably an integer from 2 to 30, preferably an integer from 2 to 20, preferably an integer from 2 to 10, preferably 2, 3, 4, 5, 6, 7, 8, 9.

[0275] In one embodiment, L 1 A cleavable sequence comprising an amino acid sequence cleavable by an enzyme, wherein the amino acid sequence cleavable by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In one embodiment, the amino acid sequence cleavable by the enzyme is selected from -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof; a preferred amino acid sequence cleavable by the enzyme is -Gly-Gly-Phe-Gly-. In one embodiment, L 1 is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably, -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof. In one embodiment, L 1 It means -Val-Cit-.

[0276] In one embodiment, Sp1 is selected from PABC (p-aminobenzyloxycarbonyl), acetal, heteroacetal and a combination thereof; preferably, Sp1 is acetal, heteroacetal or PABC; further preferably, the heteroacetal is selected from N,O-heteroacetal; more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is linked to an amino acid sequence that can be cleaved by the enzyme, U does not exist, or is CH2, O, S or NH, preferably O or S.

[0277] In one embodiment, B is absent, or is -NH-CH2-U-, or is -NH-CH2-U-(CH2) g-(CO)-, wherein g is 1, 2, 3, 4, 5 or 6, U is absent or is CH2, O, S or NH, preferably O or S. In one embodiment, B is absent. In one embodiment, B is 1), 2), or a combination of 1) and 2): 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 In one embodiment, B is -NH-CH2-U- or -NH-CH2-U-(CH2) g -(CO)-, U is absent or is CH2, O, S or NH, preferably O or S. In one embodiment, B is connected to the load via an amide bond, an ester bond or an ether bond. In one embodiment, B is selected from: (-PABC-), -NH-CH2-U-, or -NH-CH2-U-(CH2) g -(CO)-; wherein g is 1, 2, 3, 4, 5 or 6; U is absent or is CH2, O, S or NH, preferably O or S.

[0278] In one embodiment, -L 1 -B- stands for -Val-Cit-PABC-.

[0279] In one embodiment, -L 2 -L 1 -B- denotes -Gly-Gly-Gly-Val-Cit-PABC-.

[0280] In one embodiment, Ld2 and each Ld1 are independently selected from a bond, or

[0281] Each i, j and k is independently selected from an integer from 1-100.

[0282] In one embodiment, each i, j, and k is independently selected from an integer from 1 to 20. In one embodiment, each i, j, and k is independently selected from an integer from 1 to 12.

[0283] In one embodiment, each i is independently selected from an integer from 2 to 8; in one embodiment, i is about 4.

[0284] In one embodiment, each j is independently selected from an integer from 8 to 12; in one embodiment, j is approximately 8 or 12.

[0285] In one embodiment, each k is independently selected from an integer from 1 to 7; in one embodiment, k is about 1 or 3 or 5.

[0286] In one embodiment, Ld2 and each Ld1 are independently selected from a bond; or a C with an amino group and a carbonyl group at both ends. 1- 20 Alkylene, or a PEG fragment of a certain length with an amino group and a carbonyl group at both ends (represented as -(PEG) i -), or one or more natural amino acids, each of which is independently unsubstituted or substituted with a PEG fragment of a certain length (represented as -CO-(PEG) j -)replace.

[0287] In one embodiment, -(PEG) i -Contains -(O-C2H4) i -or-(C2H4-O) i -, and optionally appended with C at one end 1-10 Alkylene; -(PEG) j -Contains -(O-C2H4) j -or-(C2H4-O) j -, and optionally appended with C at one end 1- 10 In one embodiment, -(PEG) i - contains -C2H4-(O-C2H4) i -or-(C2H4-O) i -C2H4-.

[0288] 3. Immobilization of endoglycosidase

[0289] The support can be a solid form or a semisolid form made of any material. The limiting examples of the support can include, but are not limited to, resin (for example, agarose resin, silicone resin, polymethyl methacrylate resin, epoxy resin or cellulose resin), gel (such as alginate hydrogel), pearl / microsphere / particle (for example, polystyrene beads, magnetic particles), plate, hole, pipe, film, membrane, matrix and glass (for example, slide).

[0290] In some embodiments, the support is a resin. In some embodiments, the support is selected from the group consisting of: agarose resin, silicone resin, polymethyl methacrylate resin and cellulose resin. In some embodiments, the support is a highly cross-linked agarose resin.

[0291] The method of enzyme immobilization has adsorption, covalent or non-covalent binding, embedding, encapsulation and cross-linking.Expect to retain the maximum enzymatic activity of endoglycosidase after fixation and to have a minimum amount of free endoglycosidase in the conjugate product after the coupled reaction.In some embodiments, the support surface is modified to comprise one or more functional groups so that the endoglycosidase fusion protein can be covalently fixed on the support.

[0292] In some embodiments, the support comprises one or more chemically active functional groups that can form covalent bonds with reactive groups (such as amines, sulfhydryls, and carboxylates) of the endoglycosidase fusion protein or with reactive groups in the haloalkyl substrate, or the support comprises one or more binding partners of corresponding binding tags / affinity tags contained in the endoglycosidase fusion protein. The correspondence between chemically active functional groups and reactive groups, or between binding tags / affinity tags and binding partners is known in the art.

[0293] In some embodiments, the support comprises chemically active functional groups that can form covalent bonds with reactive groups on the endoglycosidase fusion protein (e.g., amines, sulfhydryls, and carboxylates) or with reactive groups in the haloalkyl substrate. In some embodiments, the support comprises functional groups selected from the group consisting of cyanates, isothiocyanates, isocyanates, carbodiimides, N-hydroxysuccinimide (NHS) esters, amines, carbonates, epoxides, maleimides, haloacetyl groups, aziridines, ethyl chloroformate, and fatty aldehydes.

[0294] In some embodiments, the support is an epoxy-activated resin, CNBr (cyanogen bromide)-activated resin or NHS-activated resin. In some embodiments, the support is an epoxy-activated resin. In some embodiments, the support is an epoxy-activated agarose resin. In some embodiments, the support is an epoxy-activated highly cross-linked agarose resin. In some embodiments, before reacting with the haloalkyl substrate, the epoxy-activated resin is pretreated to introduce an amino group. In some embodiments, the pretreatment of the epoxy-activated resin is carried out using ammonia. In some embodiments, the pretreatment of the epoxy-activated resin results in the introduction of an amino group on the oxirane ring and the ring opening of the oxirane ring provides a hydroxyl group. Such a hydroxyl group is optionally blocked in the subsequent process of preparing the support. In some embodiments, the pretreatment of the epoxy-activated resin results in the introduction of an amino group on the oxirane ring and the ring opening of the oxirane ring provides a hydroxyl group, which is optionally esterified by an esterifying agent (for example, an acetylating agent, such as Ac2O) in the subsequent process of preparing the support. Such pretreated epoxy-activated resin is within the scope of "epoxy-activated resin" as defined above. In some embodiments, the resin is an agarose resin (such as a highly cross-linked agarose resin) or a polymethyl methacrylate resin.

[0295] In some other embodiments, the support comprises one or more binding partners corresponding to the binding tag / affinity tag contained in the endoglycosidase fusion protein, such as an additional tag or affinity tag. The corresponding relationship between reactive groups or between binding tags / affinity tags and binding partners is known in the art. Examples of binding tags / affinity tags and corresponding binding partners can include, but are not limited to, His tags and Ni tags. 2+ , biotin / SPB tag / Strep tag / Strep tag II and streptavidin / avidin / neutravidin, GST tag and glutathione, Fc tag and protein A, calmodulin tag and Ca 2+ , MBP and amylose, S tag and RNase S-protein, SNAP tag and benzylguanine (BG) derivatives, and CLIP tag and benzylcytosine (BC) derivatives.

[0296] In some embodiments, the support is functionalized by comprising a haloalkyl linker to form a covalent interaction with the Halo tag. The haloalkyl linker can be introduced into the support by covalently linking one or more functional groups contained in the support to one or more reactive groups in the haloalkyl substrate, and the support thus obtained is also referred to as a haloalkyl linker-modified support. The haloalkyl linker-modified support is within the scope of the "support" defined above. Examples of haloalkyl substrates include, but are not limited to, for example, those described in US20060024808A1 and WO2006093529. For haloalkyl substrates and methods for preparing such supports, see, for example, U.S. Patent Nos. 7,429,472, 7,888,086 and 8,202,700, and the description in Japanese Patent No. 4748685, the relevant contents of which are incorporated herein by reference.

[0297] The haloalkyl substrate may comprise a haloalkyl moiety comprising a primary or secondary halide. In some embodiments, the haloalkyl substrate comprises a primary halide. The halide in the haloalkyl moiety is selected from F, Cl, Br, and I. In some embodiments, the halide in the haloalkyl moiety is selected from Cl and Br. In some embodiments, the haloalkyl substrate has the structure of Formula (V): (F1 o ―H1 e ) r ―Lh―(F2 e ―H2 o ) s (V)

[0298] in,

[0299] F1 and F2 are independently moieties comprising reactive groups that can form covalent bonds with chemically active functional groups contained on the support;

[0300] H1 and H2 are independently selected from halogenated C 2-30 alkyl;

[0301] Lh is a chemical bond or C 3-200 Alkylene, wherein one or more (-CH2-) structures in the alkylene are optionally replaced by -O-, -NH-, -(CO)-, -NH(CO)-, and -(CO)NH-;

[0302] Lh is optionally substituted with 1, 2 or 3 substituents selected from -OC 1-10 Alkyl, -NH-C 1-10 Alkyl, -(CO)-C 1-10 Alkyl, -NH(CO)-C 1-10 Alkyl and -(CO)NH-C 1-10 alkyl;

[0303] o is 0 or 1, e is 0 or 1, provided that o and e are different;

[0304] r is an integer from 1 to 100;

[0305] s is an integer from 1 to 100.

[0306] In some embodiments, r is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, s is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the reactive group in F1 or F2 is selected from amino, amine, sulfhydryl, and active ester. In some embodiments, the active ester contains one or more carboxylic acid radicals (such as in a suitable carbonate monoester of an alcohol or phenol, for example, an electron-deficient phenol such as 4-nitrophenol; or as in an NHS ester or a sulfo-NHS ester) or one or more sulfonic acid radicals (such as in a methanesulfonic acid active ester, for example, MsO-). In a specific embodiment, F1 or F2 is

[0307] In some embodiments, H1 and H2 are independently selected from halo-C 2-20 In some embodiments, H1 and H2 are independently selected from halo-C 2-10 Alkyl, in particular halogenated C6 alkyl. In some specific embodiments, the alkyl in H1 or H2 is a straight chain alkyl. In some embodiments, H1 or H2 is (CH2) 2-30 -X. In some embodiments, H1 or H2 is (CH2) 2-20 -X. In some embodiments, H1 or H2 is (CH2) 2-10 -X, in particular (CH2)6-X, wherein X is a halogen selected from F, Cl, Br and I.

[0308] In some embodiments, the support is HaloLink TM resin (Promega).

[0309] In some embodiments, the support is a resin that may include a haloalkyl linker comprising -(CH2) 2-30 -X structure, wherein X is a halogen selected from F, Cl, Br and I. In a specific embodiment, the support is a resin modified with a halogenated aryl linker. In some embodiments, the support is agarose resin or polymethyl methacrylate resin. In some embodiments, the support is a highly cross-linked agarose resin.

[0310] In some embodiments, o is 1, e is 0, r is 1, s is 1, and F1 is Lh is H2 is (CH2) 2-20 -Cl, and the haloalkyl substrate is a chloroalkyl substrate having a structure of formula (III):

[0311] wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19.

[0312] In a specific embodiment, u is 3, v is 2, and w is 5, and the chloroalkyl substrate has the structure of the following formula (III-1):

[0313] In some embodiments, the support is a chloroalkyl linker-modified support and has the structure of Formula (IV):

[0314] wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19; " represents a support, which is a resin, bead, membrane, gel, matrix, film, plate, well, tube, slide, or surface. In some embodiments, the support is a resin. In some embodiments, the support is an agarose resin, a silicone resin, a polymethyl methacrylate resin, or a cellulose resin. In some embodiments, the support is a highly cross-linked agarose resin. Note that for clarity, only a single chloroalkyl-linker moiety is described as being attached to the support, but it is understood that there may be many such chloroalkyl-linker moieties attached to the support.

[0315] In some embodiments, the chloroalkyl linker modified support of formula (IV) is represented by The invention is prepared from a resin, bead, membrane, gel, matrix, film, plate, well, tube, slide or surface and a chloroalkyl substrate of formula (III).

[0316] In some embodiments, the chloroalkyl linker-modified support as shown in formula (IV) is prepared from a pretreated epoxy-activated resin, wherein the epoxy-activated resin is prepared by introducing an amino group into the oxirane ring of the epoxy-activated resin. During the pretreatment, the ring opening of the oxirane ring provides a hydroxyl group, which is optionally esterified with Ac2O in a subsequent step of preparing the support. The support shown in formula (IV) has the structure of formula (IV-1):

[0317] in,

[0318] Substructure represents a pre-treated epoxy activated resin, wherein The moiety represents an oxirane ring, which reacts with an amino group and opens the ring to give a hydroxyl group, which is then esterified to form AcO-, The portion represents the other portion of the pre-treated epoxy activated resin.

[0319] In some embodiments, the immobilized endoglycosidase fusion protein has the following structure:

[0320] Support----Linker----Halo Tag----EndoS / EndoS2----HisTag

[0321] in,

[0322] Support is a support (such as a solid support), for example, selected from a resin, a bead, a membrane, a gel, a matrix, a film, a plate, a well, a tube, a slide, or a surface. In some embodiments, the support is a resin. In some embodiments, the support is an agarose resin, a silicone resin, a polymethyl methacrylate resin, or a cellulose resin. In some embodiments, the support is a highly cross-linked agarose resin or a polymethyl methacrylate resin. In some embodiments, the support is selected from the group consisting of an NHS-activated resin, a CNBr-activated resin, and an epoxy-activated resin;

[0323] Linker is a linker moiety covalently bound to a support, for example, a chain comprising 10-60 carbon atoms, optionally comprising one or more ether, ester, carbamate and / or amide bonds; for example, a linker moiety of formula (VI) or (VI-1)

[0324] wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19;

[0325] HaloTag is a Halo tag (haloalkane dehalogenase) covalently bound to a linker;

[0326] EndoS / EndoS2 is an endoglycosidase;

[0327] HisTag is a histidine tag;

[0328] One or more of the "----Linker----HaloTag----EndoS----HisTag" parts are bound to the same support.

[0329] 4. Coupling equipment

[0330] The coupling device disclosed in patent application WO2022170676A or CN114480115A can be used for site-specific coupling of the N-glycosylation site of the antibody Fc region and a load, specifically for the method of preparing a conjugate or an antibody-drug conjugate of the present invention, wherein the coupling device comprises a flow reactor and a fluid transport unit, wherein the flow reactor is filled with an immobilized endoglycosidase, and the fluid transport unit is connected to the inlet fluid of the flow reactor and transports a donor containing oxazoline oligosaccharides and an antibody (or a protein containing an Fc region) containing a GlcNAc motif to the flow reactor.

[0331] In some embodiments, the coupling device includes: at least one flow reactor having an inlet and an outlet, the flow reactor being filled with a medium (e.g., a matrix such as chromatographic beads, fibers, or films), and the endoglycosidase being immobilized on the medium; a fluid delivery unit being in fluid communication with the inlet of the flow reactor and being configured to continuously provide at least one reaction fluid to the flow reactor according to different stages of the coupling process, the at least one reaction fluid comprising a donor containing an oxazoline oligosaccharide and an antibody containing a GlcNAc motif (or a protein containing an Fc region); and a fluid collection unit being in fluid communication with the outlet of the flow reactor and being configured to control the collection of the fluid flowing out of the outlet of the flow reactor according to different stages of the coupling process. During the period when the at least one reaction fluid continuously flows through the flow reactor, the donor containing an oxazoline oligosaccharide and the antibody containing a GlcNAc motif (or a protein containing an Fc region) undergo a coupling reaction under the catalysis of the endoglycosidase to generate an antibody conjugate or a conjugated drug.

[0332] In some embodiments, the endoglycosidase is directionally immobilized on the medium and loaded into a flow reactor, so that when the reaction fluid flows through the flow reactor, the two reaction components of the conjugate to be generated contained in the reaction fluid are continuously and stably coupled.

[0333] Compared with chemical coupling, the coupling device of the present invention greatly reduces process steps, significantly reduces process complexity, and is very suitable for saving expensive manufacturing costs. Moreover, by using a flow reactor, the linear amplification and continuous flow production of the coupling process can be realized, to meet the industrial demand for higher output, shorten the unit coupling time and reduce the occupied space in the manufacturing area. By this coupling device, bioconjugates can be produced, the site-specific coupling between the load-connector and the antibody (or the protein containing the Fc region) can be realized, homogeneity is improved, and therefore the treatment window is widened. In addition, the coupling process can be integrated with the production process flow of biomolecules such as monoclonal antibodies. For example, coupling can be completed in the production stage of monoclonal antibody intermediates and single antigen liquids. Therefore, this process flexibility is high and has good consistency.

[0334] In some embodiments, at least one reaction fluid comprises a first reaction fluid and a second reaction fluid, wherein the first reaction fluid comprises a donor containing an oxazoline oligosaccharide or an antibody (or a protein containing an Fc region) containing a GlcNAc motif, and the second reaction fluid comprises an antibody (or a protein containing an Fc region) containing a GlcNAc motif or a donor containing an oxazoline oligosaccharide.

[0335] In some embodiments, the coupling process comprises the following stages in sequence: pre-reaction equilibration, coupling reaction, post-reaction recovery, and post-recovery flushing, and the fluid transport unit is further configured to: continuously provide a buffer to the flow reactor during the pre-reaction equilibration, post-reaction recovery, and post-recovery flushing; and continuously and simultaneously provide a donor containing an oxazoline oligosaccharide and an antibody containing a GlcNAc motif (or a protein containing an Fc region) to the flow reactor during the coupling reaction.

[0336] In certain embodiments, buffer, the first reaction fluid and the second reaction fluid are stored in the first container, the second container and the third container respectively. The fluid delivery unit includes the first transfer pump and the second transfer pump. The first container and the second container are connected to the first transfer pump via the first container outlet pipeline and the second container outlet pipeline respectively, and the third container is connected to the second transfer pump via the third container outlet pipeline. The first transfer pump and the second transfer pump are connected to the inlet main pipe via the first inlet branch pipe and the second inlet branch pipe respectively, and the inlet main pipe is connected to the inlet of the flow reactor. And, during the reaction balance, reaction post-recovery and recovery post-rinsing period, the first transfer pump sends the buffer in the first container to the inlet main pipe, and during the coupled reaction, the first transfer pump sends the first reaction fluid in the second container to the inlet main pipe, and the second transfer pump sends the second reaction fluid in the 3rd container to the inlet main pipe.

[0337] In some embodiments, the fluid delivery unit further includes a first valve, a second valve, a third valve, and a fourth valve. The first valve, the second valve, and the third valve are respectively disposed on the first container outlet pipeline, the second container outlet pipeline, and the third container outlet pipeline, and are respectively used to control the flow of fluid in the first container outlet pipeline, the second container outlet pipeline, and the third container outlet pipeline, and the fourth valve is disposed on the first inlet branch pipe, and is used to control the flow of fluid in the first inlet branch pipe.

[0338] In some embodiments, during pre-reaction equilibration, post-reaction recovery, and post-recovery flushing, the first valve and the fourth valve are open, and the second valve and the third valve are closed, and during the coupling reaction, the first valve is closed, and the second, third, and fourth valves are open.

[0339] In some embodiments, the first container outlet pipe, the second container outlet pipe, the third container outlet pipe, the first inlet branch pipe, the second inlet branch pipe, and the inlet main pipe are disposable or non-disposable and are each made of one of stainless steel, titanium, and silicone. The first container, the second container, and the third container are each selected from the group consisting of a disposable liquid storage bag, a disposable liquid storage bottle, a stainless steel container, and disposable and non-disposable glass or plastic containers.

[0340] In some embodiments, the fluid collection unit is further configured to: collect the fluid flowing out of the outlet of the flow reactor into a fourth container during pre-reaction equilibration and post-recovery flushing; and collect the fluid flowing out of the outlet of the flow reactor into a fifth container during the coupling reaction and post-reaction recovery.

[0341] In some embodiments, the fourth container and the fifth container are connected to the outlet main pipe via the fourth container inlet pipe and the fifth container inlet pipe, respectively, the outlet main pipe is connected to the outlet of the flow reactor, and the fluid collection unit includes a fifth valve and a sixth valve, which are respectively arranged on the fourth container inlet pipe and the fifth container inlet pipe, and are respectively used to control the fluid flow in the fourth container inlet pipe and the fifth container inlet pipe.

[0342] In some embodiments, during pre-reaction equilibration and post-recovery flushing, the fifth valve is open and the sixth valve is closed, and during the coupling reaction and post-reaction recovery, the fifth valve is closed and the sixth valve is open.

[0343] In some embodiments, the fourth container inlet line and the fifth container inlet line are disposable or non-disposable and are respectively made of one of stainless steel, titanium, and silicone. The fourth container and the fifth container are respectively selected from one of the following: a disposable liquid storage bag, a disposable liquid storage bottle, a stainless steel container, and a disposable and non-disposable glass or plastic container.

[0344] In some embodiments, the coupling device further comprises: a temperature control unit configured to control the temperature of the fluid flowing into the inlet of the flow reactor and the fluid flowing out of the outlet of the flow reactor during the coupling process.

[0345] In some embodiments, the temperature control unit includes: a heating module, which is arranged at the inlet of the flow reactor and is used to heat the fluid flowing into the inlet; and a cooling module, which is arranged at the outlet of the flow reactor and is used to cool the fluid flowing out of the outlet.

[0346] In some embodiments, the coupling device further includes: a sampling and detection unit, which is connected to the outlet fluid of the flow reactor and is configured to: collect sample fluid from the fluid flowing out of the outlet of the flow reactor according to a preset sampling time; and detect the conjugate in the sample fluid to obtain a detection result, which indicates whether the conjugate meets the preset standard.

[0347] In some embodiments, the sampling and detection unit includes a sampling pump, a first switching valve, an elution pump, at least one analytical column, and a detector. The sampling pump is connected to the outlet of the flow reactor via a sampling line, the first switching valve is provided with a sample loop, and the first switching valve switches between a first state and a second state according to a preset sampling time. When the first switching valve is in the first state, the sampling pump is in fluid communication with the sample loop, and collects a sample fluid from the fluid flowing out of the outlet of the flow reactor via the sampling line and pumps it into the sample loop. When the first switching valve is in the second state, the elution pump, the sample loop, the at least one analytical column, and the detector are in fluid communication via the detection line, and the elution pump pumps the eluate into the detection line, causing the eluate to flow through the sample loop, thereby driving the sample fluid in the sample loop to flow through one of the at least one analytical column and then into the detector.

[0348] In some embodiments, there are two analytical columns, and the sampling and detection unit further includes a second switching valve and a wash pump, wherein the second switching valve switches between two states. When the second switching valve is in either state, the sample loop and the detector are in fluidic communication with one of the two analytical columns, and the eluent drives the sample fluid in the sample loop into the analytical column. Furthermore, the wash pump is in fluidic communication with the other analytical column and pumps buffer into the other analytical column to balance the other analytical column.

[0349] In some embodiments, the first switching valve is a six-port valve, the second switching valve is a ten-port valve, and the elution pump is a quaternary pump.

[0350] In some embodiments, the coupling device further comprises a recycling unit disposed between the inlet and outlet of the flow reactor. When the test result indicates that the conjugate does not meet a preset standard, the fluid collection unit is configured to stop collecting the fluid flowing out of the outlet of the flow reactor, and the recycling unit is configured to control the fluid flowing out of the outlet of the flow reactor to re-enter the inlet to conduct the coupling reaction in the flow reactor again.

[0351] In some embodiments, the recycling unit includes a seventh valve disposed on a recycling line. The recycling line is connected between the inlet and outlet of the flow reactor, and a recycling container is disposed on the recycling line. When the test results indicate that the conjugate does not meet a predetermined standard, the seventh valve opens, and fluid flowing out of the outlet of the flow reactor flows through the recycling line and the recycling container before flowing into the inlet.

[0352] In some embodiments, the flow reactor is a coupled column (ie, a prepacked column).

[0353] In some embodiments, the coupling device further comprises at least one selected from the group consisting of a pressure sensing module, a flow detection module, a pH metering module, a conductivity metering module, and a UV detection module. In some embodiments, the pressure sensing module, the flow detection module, the pH metering module, the conductivity metering module, and the UV detection module can be disposed at the inlet and / or outlet, respectively.

[0354] The full text of patent application WO2022170676A or CN114480115A is incorporated into this application. DETAILED DESCRIPTION

[0355] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0356] Instruments, materials and reagents

[0357] Unless otherwise specified, the instruments and reagents used in the examples are commercially available and can be used directly without further purification.

[0358] Ni Sepharose 6 FF: Metal chelate affinity chromatography medium, also known as fixed metal ion affinity chromatography, its principle is to use some amino acids on the surface of proteins, such as histidine, tryptophan, cysteine, etc., to bind to metal ions (Cu 2+ , Zn 2+ , Ni 2+ ,Co 2+ , Fe 3+) to separate proteins. These interactions include coordinate bonding, electrostatic adsorption, and covalent bonding, with coordinate bonding being the most common. Among these, the hexahistidine tag (His-Tag) is the most widely used.

[0359] Q Sepharose FF / Capto S impact: The separation of molecules by ion exchange is based on the difference in surface electrostatic charge. Proteins are composed of many different amino acids containing weak acid and weak base groups, and the surface electrostatic charge will gradually change with the change of the surrounding pH, that is, the protein molecules are amphiphilic molecules. During the ion exchange chromatography separation process, the binding and elution of specific molecules are achieved by controlling the reversible interaction between the charged molecules and the ion exchange fillers with opposite charges, thereby achieving the separation effect. The surface electrostatic charge of proteins in an environment with the same pH value as the isoelectric point is zero and will not interact with the charged fillers. When the pH value of the environment is higher than its isoelectric point, the protein will bind to the positively charged filler, that is, the anion exchanger; and when the pH value of the environment is lower than its isoelectric point, the protein will bind to the negatively charged filler, that is, the cation exchanger.

[0360] SDS-PGAE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS is an anionic detergent that acts as a denaturant and cosolvent, breaking intra- and intermolecular hydrogen bonds, removing hydrophobic interactions, causing molecular unfolding, and disrupting the secondary and tertiary structures of proteins. SDS binds to denatured proteins, causing them to become negatively charged. The amount of SDS bound is proportional to molecular weight. At saturation, each gram of protein can bind 1.4 g of SDS. This negative charge significantly exceeds the original charge of the protein, eliminating charge and structural differences between molecules. Therefore, the mobility of SDS-protein complexes in acrylamide gel electrophoresis is related solely to protein size. Strong reducing agents (such as β-mercaptoethanol and dithiothreitol) can break intra- and intermolecular disulfide bonds, making protein separation more efficient.

[0361] Example 1. Preparation of endoglycosidase fusion protein

[0362] 1.1 Amino acid sequence of Halo-EndoS2-His (Halo is single-underlined, His Tag is italicized, and GGGGSGGGGS is the linker sequence)

[0363] 1.2 Halo-Endo S2-His cloning

[0364] The nucleic acid sequence encoding Halo-Endo S2-His was synthesized using standard gene synthesis methods and inserted into the pET expression vector. The Halo-Endo S2-His expression plasmid was transformed into Escherichia coli BL21(DE3). The cells were incubated in LB (Luria-Bertani) medium supplemented with the appropriate antibiotics and cultured at 37°C until the OD600 reached 0.5-1.0.

[0365] 1.3 Halo-Endo S2-His Tag Purification (Reactor)

[0366] Remove the glycerol bacteria and inoculate them into LB liquid medium containing the corresponding antibiotics for culture. Cultivate at 100-300 rpm and 37°C with shaking for 2-8 hours. When the OD600 reaches 0.5-1.0, inoculate it into a 10L reactor and culture for 5-10 hours. Add IPTG at a final concentration of 0.2mM and induce expression at 16°C overnight. Collect the bacterial pellet by centrifugation at 2-8°C for 10-30 minutes at a speed of 3000-5000 rpm. Add the equilibration buffer to resuspend the cells and crush them under high pressure. Centrifuge at 2-8°C at 5000-10,000 rpm for 10-60 minutes to separate the supernatant and precipitate.

[0367] For Ni column affinity purification, connect the Ni column to a protein purification system and rinse thoroughly with purified water and equilibration buffer. Load the supernatant at a low flow rate. Wash with equilibration buffer and 80 mM imidazole buffer, respectively. Elution is then performed with 500 mM imidazole buffer. Analyze by SDS-PAGE.

[0368] As shown in Figure 1, Halo-Endo S2-His is attached to the Ni magnetic beads, and the target sample is absent in the flowthrough. Halo-Endo S2-His is eluted at 500 mM imidazole. This result demonstrates that Halo-Endo S2-His can be scaled up using a reactor and linearly scaled up using a Ni chromatography column.

[0369] Example 2. Preparation of immobilized Halo-Endo S2-His

[0370] Preparation process

[0371] 2.1 Preparation of Chloroalkyl-Linker-Modified Resin (Chloro Resin)

[0372] Methods for preparing chlorine resins are described, for example, in US Patent Nos. 7,429,472, 7,888,086, and 8,202,700, the entire contents of which are incorporated herein by reference. The resins used to prepare the chlorine resins are shown in Table 1.

[0373] Table 1. Resins

[0374] method:

[0375] (1) Pretreatment

[0376] For NHS-activated resin (Bestchrom) and CNBr-activated resin (Bestchrom):

[0377] Filter the resin with isopropanol, wash the filter cake once with DMF (N,N-dimethylformamide), and then vacuum dry. Transfer the filter cake to a flask with DMF and stir. Next, add ethylenediamine to the mixture and stir for 10 to 15 hours. Filter and wash the filter cake with DMF. Drain the liquid.

[0378] For epoxy activated resins:

[0379] Filter the resin with isopropanol, wash the filter cake once with H2O, and then suck dry. Transfer the filter cake to a flask and stir. Next, add 25% to 28% concentrated aqueous ammonia to the mixture, slowly heat the system to 40-50°C, and react with stirring at 40-50°C. Lower the system temperature to 20-30°C and filter the mixture. Wash the filter cake with H2O until the pH of the filtrate reaches about 7-8. Next, wash the filter cake with DMF and drain the liquid;

[0380] (2) Transfer the filter cake from step (1) to a flask and stir. Next, add DMF and triethylamine containing the chloroalkyl substrate of formula (III-1) to the system in sequence. Stir and react. Next, filter the system and wash the filter cake with DMF, and finally drain the liquid.

[0381] (3) Transfer the filter cake from step (2) to a flask. Turn on stirring. Next, add Ac2O and triethylamine to the mixture in sequence. Stir the reaction. Then, filter the mixture and wash the filter cake with DMF. Then wash the filter cake with H2O and drain the liquid. Finally, transfer the mixture to a container with 20% ethanol for storage.

[0382] Result: A chlorine resin having the structure of formula (IV-1) is obtained:

[0383] in It is a highly cross-linked agarose resin or polymethyl methacrylate resin.

[0384] Halo-Endo S2-His was mixed with Chloro resin and incubated at room temperature for 10 minutes to 24 hours. The washing step was repeated three times with 20mM Tris-HCl, 150mM NaCl, pH 6.0-10.0 buffer. The immobilized endoglycosidase fusion protein (Halo-Endo S2-His covalently linked to Chloro resin) was assayed for activity. If the protein passed the assay, it was washed with 20mM Tris-HCl, 150mM NaCl and stored at 2-8°C.

[0385] 2.2 Immobilized enzyme loading

[0386] 1) Take 250 μl of each chlororesin to be tested, add excess glycosidase, place on a rotary mixer, and immobilize at room temperature for 2 hours;

[0387] 2) During the Halo-Endo S2-His immobilization process, the immobilized enzyme supernatant was collected at 15 min, 30 min, 1 h, and 2 h, centrifuged at 3000 g for 3 min at room temperature each time, and the supernatant was aspirated and the enzyme concentration in the supernatant was measured using a Nanodrop UV spectrophotometer;

[0388] 3) Calculate the concentration of endoglycosidase at each time point, subtract the concentration at each time point from the initial enzyme concentration, calculate the immobilized enzyme amount at the time of timing, and draw a loading curve of Halo-Endo S2-His immobilized on the medium.

[0389] SDS-PAGE was used to detect the trend of protein changes in the supernatant, as shown in Figure 2. The results showed that as time went on, the amount of Halo-Endo S2-His in the supernatant became less and less, and it was specifically fixed to the chlorinated resin.

[0390] Example 3. Preparation of linker-load

[0391] 3.1 Preparation of disaccharide substrates

[0392] Preparation of disaccharide substrate compound 1

[0393] The following steps were used to prepare compound 1, whose structure is as follows:

[0394] (1) Preparation of compound 1c

[0395] Under oil pump vacuum conditions, a 100mL Schlenk reaction flask was baked with a heat gun for 5 minutes. After cooling, activated molecular sieves were added to the system and baked for another 5 minutes. Vacuum and replace nitrogen three times. Under nitrogen protection, compound 1a (4.41g, 7.96mmol) was added to the system. After stirring for 3 minutes, anhydrous dichloromethane (30mL) was added and stirred for 0.5 hours. In addition, following the same operation, compound 1b (1.89g, 3.98mmol, dissolved in 20mL of anhydrous dichloromethane) was added to another 50mL Schlenk reaction flask to which activated molecular sieves had been added. Stir for 1 hour and pre-dry to remove residual moisture in the system.

[0396] Under nitrogen, 1-(phenylsulfinyl)piperidine (BSP, 1.37 g, 6.56 mmol) and 2,4,6-tri-tert-butylpyrimidine (TTBP, 2.94 g, 11.94 mmol) were added to the dried solution of compound 1a at room temperature and stirred for an additional 20 minutes. The reaction flask was placed in a dry ice / ethyl acetate bath and cooled to -65°C. Trifluoromethanesulfonic anhydride (1.2 mL, 7.16 mmol) was then added. Two minutes later, a pre-dried dichloromethane solution of compound 1b was added. The resulting reaction mixture was stirred at -65°C until the reaction was complete as determined by TLC (developing solvent: EtOAc / PE = 1 / 8) (approximately 3 hours). Saturated sodium bicarbonate solution was added to the system to quench the reaction, and the liquid was extracted with dichloromethane (150 mL × 3). The combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The product was wet-loaded and separated by column chromatography (eluent: EtOAc / PE = 1 / 12-1 / 10) to obtain compound 1c (2.72 g, yield 75.4%, colorless viscous oily liquid). 1 H NMR (400 MHz, chloroform-d) δ 7.55 (dd, J = 7.6, 2.1 Hz, 2H), 7.51–7.28 (m, 28H), 5.60 (s, 1H), 5.14 (d, J = 10.4 Hz, 1H), 5.00 (d, J = 12.0 Hz, 1H), 4.94 (d, J = 11.8 Hz, 1H), 4.90–4.80 (m, 2H), 4.76 (d, J = 12.0 Hz, 1H), 4.74–4.63 (m, 3H), 4.57 (s, 1H, H 1’ ),4.48(d,J=12.1Hz,1H),4.37(d,J=8.1Hz,1H,H 1),4.22–4.10(m,2H),4.05(t,J=9.3Hz,1H),3.80(d,J=3.1Hz,1H),3.72(dd,J=11.2,2.2Hz,1H),3.67–3.52(m,3 H), 3.49 (dd, J = 9.8, 3.1Hz, 1H), 3.41 (t, J = 9.3Hz, 1H), 3.35 (dt, J = 9.8, 2.9Hz, 1H), 3.16 (td, J = 9.7, 4.8Hz, 1H). C 54 H 56 N3O 10 + [M+H] + MS (ESI) m / z, calculated value 906.4, found value 906.7.

[0397] (2) Preparation of compound 1d

[0398] To a 25 mL Schlenk reaction flask at room temperature, compound 1c (228 mg, 0.252 mmol), chloroform (1.2 mL), pyridine (1 mL), and AcSH (1.2 mL) were added sequentially to dissolve the mixture. The flask was sealed with a stopper and stirred at 60°C until the reaction was nearly complete as determined by HPLC, which took approximately 18 hours. After concentration under reduced pressure to remove most of the solvent, ethyl acetate (50 mL) was added to the reaction. The mixture was washed sequentially with saturated sodium bicarbonate solution (30 mL), 1 M hydrochloric acid (10 mL x 4), and saturated sodium bicarbonate solution (30 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 10 to 1 / 1) to afford compound 1d (200 mg, 86% yield, as a white solid). 1 H NMR (400 MHz, chloroform-d) δ7.57–7.49 (m, 2H), 7.48–7.39 (m, 5H), 7.39–7.24 (m, 23H), 5.80 (d, J = 8.1 Hz, 1H), 5.59 (s, 1H), 5.00 (d, J = 6.7 Hz, 1H), 4.97–4.87 (m, 3H), 4.87–4.75 (m, 2H), 4.69–4.59 (m, 4H), 4 .57(s,1H),4.45(d,J=12.0Hz,1H),4.19–4.05(m,3H),3.96(t,J=7.3Hz,1H),3.85–3.76(m,2H),3 .73–3.59(m,4H),3.49(dd,J=9.8,3.1Hz,1H),3.18(td,J=9.7,4.8Hz,1H),1.77(d,J=1.2Hz,3H).C 56 H60 NO 11 + [M+H] + MS (ESI) m / z, calculated value 922.4, found value 922.4.

[0399] (3) Preparation of compound 1e

[0400] To a 100 mL single-necked flask at room temperature, compound 1d (168 mg, 0.168 mmol), p-toluenesulfonic acid monohydrate (35 mg, 0.168 mmol), methanol (5 mL), and tetrahydrofuran (5 mL) were added sequentially. The nitrogen atmosphere was replaced three times, and the reaction was stirred at room temperature overnight until complete as determined by HPLC. The reaction system was quenched with saturated sodium bicarbonate solution (20 mL), and the liquids were extracted with dichloromethane (20 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. After purification by column chromatography (eluent: MeOH / DCM = 1 / 20), compound 1e (136 mg, 89% yield) was obtained as a white solid. 1 H NMR (400MHz, chloroform-d) δ7.43–7.24(m,29H),5.76(d,J=7.9Hz,1H),5.01(d,J=6.9Hz,1H),4.9 4(dd,J=11.8,2.3Hz,2H),4.85(d,J=11.7Hz,1H),4.77(d,J=11.8Hz,1H),4.70–4.59(m,3H ),4.58–4.47(m,3H),4.36(d,J=11.7Hz,1H),4.20(t,J=7.9Hz,1H),3.94(t,J=7.5Hz,1H) ,3.91–3.80(m,3H),3.80–3.71(m,3H),3.63–3.50(m,2H),3.22–3.14(m,2H),1.78(s,3).C 49 H 56 NO 11 + [M+H] + MS (ESI) m / z, calculated value 834.4, found value 834.5.

[0401] (4) Preparation of Compound 1f

[0402] To a 100 mL single-necked flask at room temperature, compound 1e (130 mg, 0.156 mmol), dichloromethane, tert-butanol, and water were added sequentially. Iodophenyl diacetic acid (0.5–10 equiv) and 2,2,6,6-tetramethyl-1-piperidinyl oxide (0.01–1 equiv) were added, and the reaction was stirred at room temperature overnight until complete as determined by TLC. The resulting mixture was extracted with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford compound 1f (100 mg, 75.1% yield, as a white solid). 1 H NMR (400MHz, chloroform-d) δ7.42–7.31(m,18H),7.30–7.27(m,2H),7.25–7.20(m,3H),7.15(dd,J=6.7,2.9Hz,2H),6.06(d,J= 8.3Hz,1H),4.96(d,J=6.6Hz,1H),4.93(d,J=11.9Hz,1H),4.86(d,J=11.1Hz,1H),4.81(d,J=12.1Hz,1H),4.73(d,J=11 .1Hz,1H),4.71–4.65(m,3H),4.65–4.57(m,3H),4.49(s,1H),4.46(d,J=12.0Hz,1H),4.23(t,J=7.1Hz,1H),4.15(t,J= 9.5Hz,1H),3.89(t,J=6.5Hz,1H),3.86–3.65(m,6H),3.54(d,J=9.7Hz,1H),3.30(dd,J=9.3,2.8Hz,1H),1.65(s,3H).C 49 H 52 NO 12 - [MH + ] - MS (ESI) m / z, calculated value 846.3, found value 846.3.

[0403] (5) Preparation of Compound 1

[0404] At room temperature, compound 1f (160 mg, 0.189 mmol), tetrahydrofuran, methanol, and palladium-carbon catalyst were added sequentially to a 50 mL single-necked flask. The reaction system was stirred under a hydrogen atmosphere until all the starting material disappeared as determined by TLC. The mixture was filtered, concentrated under reduced pressure, and pumped dry to obtain compound 1 (75 mg, 100% yield, as a white solid). 14 H 22 NO 12 - [MH + ] -MS (ESI) m / z, calculated value 396.1, found value 396.1.

[0405] Preparation of disaccharide substrate compound 2

[0406] The following steps were used to prepare compound 2, the structure of which is as follows:

[0407] (1) Synthesis of Compound 2-1

[0408] Step A: Synthesis of compound 2-1b

[0409] Compound 2-1a (1.0 equivalent, commercially available, CAS: 959153-39-0), MeOH, and sodium methoxide (0.1 equivalent, 5 mol / L in MeOH) were added to a single-necked flask and stirred at room temperature under a nitrogen atmosphere. The reaction progress was monitored by TLC. After the reaction was completed, 1 M hydrochloric acid was added to quench the reaction and adjust the reaction to neutrality. The solvent was removed under reduced pressure, and a small amount of toluene was added. The water was removed by azeotropic effect under reduced pressure rotary evaporation to obtain the crude product 2-1b as a light yellow viscous oily liquid, which was used directly in the next reaction without further purification.

[0410] Step B: Synthesis of compound 2-1c

[0411] To a single-necked flask, crude product 2-1b (1 equivalent), acetonitrile, camphorsulfonic acid (0.1 equivalent), and benzaldehyde dimethyl acetal (4 equivalents) were added sequentially, and the reaction system was stirred at room temperature overnight. TLC monitored the reaction progress. After completion (approximately 24 hours), saturated sodium bicarbonate solution was added to the system to quench the reaction. The liquid was extracted with ethyl acetate, and the combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by wet-loading silica gel column chromatography (eluent: PE / EtOAc = 5:1) to obtain 2-1c (white solid, 83% yield). 1H NMR (400MHz, chloroform-d) δ7.51-7.49(m,2H,Ar-H),7.45(d,J=8.0Hz,2H,Ar-H),7.42-7.37(m,5H,Ar-H),7 .36-7.31(m,3H,Ar-H),7.15(d,J=8.0Hz,2H,Ar-H),5.56(s,1H,PhCH),4.97(d,J=11.6Hz,1H,PhCH2 ),4.81(d,J=11.6Hz,1H,PhCH2),4.58(d,J=9.6Hz,1H),4.40(dd,J=10.2,4.8Hz,1H),3.80(dd,J=10 .0,10.4Hz,1H),3.70(dd,J=9.2,9.2Hz,1H),3.65(dd,J=9.2,9.2Hz,1H),3.53-3.48(m,2H),2.64(br s,1H,-OH),2.36(s,3H).C 27 H 29 O5S + [M+H] + The MS (ESI) m / z was calculated to be 465.2, and the actual value was 465.3. The NMR data were consistent with those reported in the literature (Nature 2007, 446, 896 for compound 2b1).

[0412] Step C: Synthesis of compound 2-1d

[0413] To a single-necked flask were added 2-1c (1 equivalent), dichloromethane, and triethylamine (5 equivalents) in sequence. The system was cooled to 0°C, acetic anhydride (2 equivalents) was added, and after stirring for 10 min, the reaction system was warmed to room temperature and stirred. The reaction progress was monitored by TLC. After the reaction was completed (about 6 h), saturated sodium bicarbonate solution was added to the system to quench the reaction. The liquid was extracted with dichloromethane, and the combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by wet-loading column chromatography (eluent: PE / EtOAc = 5:1) to give 2-1d (white solid, 89% yield). 1H NMR (400MHz, chloroform-d) δ7.51-7.48(m,2H,Ar-H),7.43-7.36(m,5H,Ar-H),7.34-7.25(m,5H,Ar-H),7.13(d,J =8.0Hz,2H,Ar-H),5.58(s,1H,PhCH),5.00(dd,J=8.0,8.0Hz,1H,H-2),4.87(d,J=12.0Hz,1H,PhCH2),4. 67(d,J=12.0Hz,1H,PhCH2),4.64(d,J=10.4Hz,1H,H-1),4.39(dd,J=10.8Hz,J=5.2Hz,1H),3.81(t,J=10 .4Hz,1H),3.78-3.70(m,2H),3.48(ddd,J=9.6,5.4,10.0Hz,1H),2.35(s,3H,Ar-CH3),2.05(s,3H,OAc). C 29 H 31 O6S + [M+H] + The MS (ESI) m / z was calculated to be 507.2, found to be 507.4. The NMR data were consistent with those reported in the literature for compound 3b1 in Nature 2007, 446, 896.

[0414] Step D: Synthesis of compound 2-1e

[0415] Under nitrogen atmosphere, compound 2-1d (1 equivalent), dichloromethane, and borane tetrahydrofuran (10 equivalents, 1M in THF) were added to a 100 mL two-necked flask in sequence. The system was cooled to 0°C, and dibutylboron trifluoromethanesulfonate (1.4 equivalents, 1.0M in DCM) was added dropwise. The temperature was maintained at 0°C until TLC showed that the reaction was complete (about 5 hours). Subsequently, triethylamine solution was added to the system at 0°C to quench the dibutylboron trifluoromethanesulfonate in the system, and methanol was slowly added dropwise to quench the borane tetrahydrofuran. After the system no longer produced a large amount of bubbles, water was added to ensure sufficient quenching, and then the liquid was extracted with ethyl acetate. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-loaded, and flash column chromatography (eluent: eluent: PE / EtOAc = 4:1) gave the crude product 2-1e (white solid). LCMS confirmed the structure of the product, C 29 H 33 O6S + [M+H] + The MS (ESI) m / z was calculated to be 509.2 and the actual value was 509.3. The crude product was used directly in the next reaction without fine purification.

[0416] Step E / F: Synthesis of compound 2-1f

[0417] Step E: 2-1e (1 equivalent), iodobenzene acetate (3 equivalents), TEMPO (0.5 equivalents) and tert-butanol / dichloromethane / water (volume ratio 4:4:1) were added to a single-necked flask in sequence. The resulting mixture was stirred at room temperature and the reaction progress was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1, containing 1 v / v% acetic acid). After the reaction was complete, saturated sodium thiosulfate was added to the system to quench the reaction. The mixture was extracted with dichloromethane and the combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting carboxylic acid intermediate was used directly in the next reaction without further purification.

[0418] Step F: The carboxylic acid intermediate obtained in the previous step was dissolved in DMF, and iodomethane (3 equivalents) and potassium carbonate (5 equivalents) were added. The mixture was stirred at room temperature and the reaction progress was monitored by TLC (developing solvent: PE / EtOAc = 5 / 1). After the reaction was complete (about 2 hours), water was added to the system, and the liquid was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: PE / EtOAc = 7:1) to obtain compound 2-1f (white solid, total yield of three steps 59%). 1 H NMR(400MHz, chloroform-d)δ,7.42-7.26(m,12H,Ar-H),7.16(d,J=7.6Hz,2H,Ar-H),5.02(dd,J=8.4,8 .0Hz,1H),4.83(d,J=11.6Hz,1H,PhCH2),4.79(d,J=11.2Hz,1H,PhCH2),4.70(d,J=11.6Hz,1H ),4.64(d,J=11.2Hz,1H),4.62(d,J=10.0Hz,1H),3.97(d,J=10.0Hz,1H),3.90(dd,J=8.4,8.0 Hz, 1H), 3.79 (s, 3H, OMe), 3.72 (dd, J = 8.8, 8.8Hz, 1H), 2.38 (s, 3H, Ar-CH3), 2.04 (s, 3H, OAc). C 30 H 33 O7S + [M+H] + MS (ESI) m / z, calculated value 537.2, found value 537.4.

[0419] Step G: Synthesis of compound 2-1g

[0420] To a 50 mL single-necked flask, 2-1f (1.0 equivalent) and acetone were added sequentially. The system was cooled to 0°C, and N-bromosuccinimide (1.4 equivalents) was added. Stirring was continued at 0°C for approximately 1 hour. TLC (developing solvent: PE / EtOAc = 4 / 1) indicated that the reaction was complete. Saturated sodium thiosulfate solution was added to the system to quench the reaction. The acetone was removed by rotary evaporation under reduced pressure, and water was added. Extraction was then performed with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: PE / EtOAc = 6:1) to obtain compound 2-1g (white solid, 90% yield). 23 H 27 O8 + [M+H] + MS (ESI) m / z, calculated value 431.2, found value 431.5.

[0421] Step H: Synthesis of compound 2-1

[0422] Under a nitrogen atmosphere, 2-1 g (1 equivalent), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.1 equivalent), and dichloromethane were added sequentially to a 50 mL single-necked bottle. The system was cooled to 0°C and stirred evenly. Trichloroacetonitrile (4 equivalents) was then added. The ice-water bath was removed and the temperature was naturally raised to room temperature and stirred. The reaction was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1). After the reaction was complete (approximately 2 hours), the solvent was removed by rotary evaporation under reduced pressure and purified by wet-loading column chromatography (eluent: PE / EtOAc = 4:1) to obtain compound 2-1 (a light yellow viscous oily liquid, yield 87%). 23 H 25 O7 + [M-Cl3CC(NH)O - ] + MS (ESI) m / z, calculated value 413.2, found value 413.2.

[0423] (2) Synthesis of Compound 2-2

[0424] Step A: Synthesis of compound 2-2a

[0425] 2-2a (CAS: 1235137-45-7) is commercially available and was prepared by referring to the synthesis method of compound 2 in Carbohydr Res 2016, 426, 33.

[0426] At room temperature, compound 2-2a (1 equivalent), methanol and sodium methoxide (0.1 equivalent, 5M in MeOH) were added to a single-necked flask and stirred at room temperature. The reaction was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1). After the reaction was complete (about 1 hour), dilute hydrochloric acid (1M) was added to neutralize the solution to pH = 7. After the reaction solution was concentrated, toluene was added and the residual water in the system was removed by rotary evaporation under reduced pressure using the azeotropic effect to obtain a light brown oily crude product 2-2b, which was used directly in the next reaction without purification.

[0427] Step B: Synthesis of compound 2-2c

[0428] At room temperature, to the crude product 2-2b (1 equivalent) of the previous step reaction, monohydrated p-toluenesulfonic acid (0.2 equivalent) and anhydrous acetonitrile were added in sequence and stirred thoroughly. The system was replaced with nitrogen atmosphere, and benzaldehyde dimethyl acetal (5 equivalents) was added. The resulting reaction liquid was stirred at room temperature overnight until TLC (developing solvent: PE / EtOAc = 5 / 1) showed that the reaction was complete. Then, saturated sodium carbonate solution was added to the reaction system to quench the reaction, and the liquid was extracted with dichloromethane. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-loaded, and purified by silica gel column chromatography (eluent: PE / EtOAc = 5 / 1) to give compound 2-2c (white solid, total yield of two steps 85%). 1 H NMR (400MHz, chloroform-d) δ7.63–7.54(m,2H),7.52–7.39(m,5H),7.25–7.14(m,2H),5.60(s,1H),5.53(d,J=5.4Hz,1H),4.52–4.39(m,1H),4. 28(dd,J=10.4,4.9Hz,1H),4.07(t,J=9.5Hz,1H),3.92(dd,J=10.0,5.6Hz,1H),3.79(t,J=10.3Hz,1H),3.60(t,J=9.3Hz,1H),3.08(br s,1H),2.40(s,3H).C 20 H 22 N3O4S + [M+H] + The MS (ESI) m / z was calculated to be 400.1 and found to be 399.9. The NMR data were consistent with those reported in the literature, see compound 47 in Angew. Chem. Int. Ed. 2021, 60, 12413.

[0429] Step C: Synthesis of compound 2-2d

[0430] Under nitrogen atmosphere, compound 2-2c (1 equivalent) and anhydrous tetrahydrofuran (reaction concentration 0.2 M) were added sequentially to a dry two-necked flask. The mixture was placed in an ice bath and cooled to 0°C. Sodium hydride (1.2 equivalents, 60% content, dispersed in mineral oil) was added. After the addition, the ice bath was removed and the mixture was warmed to room temperature with stirring. After 1 hour, tetrabutylammonium iodide (0.1 equivalent) and benzyl bromide (1.5 equivalents) were added. The resulting reaction system was stirred at room temperature until TLC (developing solvent: PE / EtOAc = 8 / 1) showed that the reaction was complete (about 6 hours). After completion of the reaction, water was added dropwise to quench the reaction. The liquid was extracted with ethyl acetate, and the combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: PE / EtOAc = 5 / 1) to give compound 2-2d (white solid, yield 97%). 1 H NMR (400MHz, chloroform-d) δ7.63–7.55(m,2H),7.53–7.33(m,10H),7.20(d,J=8.4Hz,2H),5.67(s, 1H),5.56(d,J=4.6Hz,1H),5.05(d,J=10.9Hz,1H),4.90(d,J=10.9Hz,1H),4.59–4.47( m,1H),4.31(dd,J=10.4,4.9Hz,1H),4.10–3.97(m,2H),3.90–3.77(m,2H),2.41(s,3H). C 27 H 28 N3O4S + [M+H] + The MS (ESI) m / z was calculated to be 490.2 and found to be 490.5. The NMR data were consistent with those reported in the literature (see compound 20 in Bioorg. Med. Chem. 2011, 19, 30).

[0431] Step D: Synthesis of compound 2-2e

[0432] Compound 2-2d (1 equivalent), tetrahydrofuran / methanol (v / v = 1:1, reaction concentration 0.5 M), and p-toluenesulfonic acid monohydrate (0.2 equivalent) were added to a single-necked flask and stirred overnight at room temperature. The reaction progress was monitored by TLC (developing solvent: PE / EtOAc = 8 / 1). After completion (approximately 12 hours), the reaction was quenched by addition of saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate and the combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and wet-loaded. Compound 2-2e was then purified by silica gel column chromatography (eluent: EtOAc / PE = 1 / 1) to afford compound 2-2e. 1H NMR (400 MHz, chloroform-d) δ 7.41–7.34 (m, 7H), 7.14 (d, J = 8.0 Hz, 2H), 5.49 (d, J = 5.2 Hz, 1H), 5.01 (d, J = 11.2 Hz, 1H), 4.77 (d, J = 11.2 Hz, 1H), 4.24–4.20 (m, 1H), 3.89–3.85 (m, 1H), 3.79–3.77 (m, 2H), 3.69–3.65 (m, 2H), 2.42 (br s, 1H), 2.34 (s, 3H). 20 H 27 N4O4S + [M+NH4] + MS (ESI) m / z, calculated value 419.2, found value 419.2.

[0433] Step E: Synthesis of compound 2-2

[0434] Compound 2-2e (1 equivalent), dichloromethane (reaction concentration 0.5 M), imidazole (2 equivalents), and tert-butyldiphenylsilyl chloride (1.5 equivalents) were added to a single-necked flask in sequence and stirred at room temperature. The reaction progress was monitored by TLC (developing solvent: EtOAc / PE = 1 / 12). After the reaction was completed (about 6 hours), saturated ammonium chloride solution was added to the system to quench the reaction, and the mixture was extracted with dichloromethane. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-loaded, and purified by silica gel column chromatography (eluent: PE / EtOAc / DCM = 15 / 1 / 1) to give compound 2-2 (yellow viscous oily liquid, yield 66%). 1 H NMR (400MHz, chloroform-d) δ7.73-7.68(m,4H),7.48-7.35(m,13H),7.06(d,J=7.6 Hz,1H),5.49(d,J=5.2Hz,1H),4.94(ABq,J=10.8Hz,2H),4.32-4.27(m,1H ),3.96-3.90(m,2H),3.87(dd,J=4.8,5.2Hz,1H),3.82(dt,J=2.8,8.8Hz, 1H),3.74-3.69(m,1H),2.70(d,J=2.8Hz,OH),2.33(s,3H),1.09(s,9H).C 36 H 42 N3O4SSi + [M+H] + MS (ESI) m / z, calculated value 640.3, found value 640.2.

[0435] (3) Synthesis of Compound 2

[0436] Step A: Synthesis of compound 2a

[0437] Add pre-activated molecular sieves to a dry two-necked flask, bake with a heat gun under oil pump vacuum conditions, cool naturally, replace nitrogen, repeat the above vacuum-exchange nitrogen operation three times, then add compound 2-1 (2 equivalents), compound 2-2 (1 equivalent) and dry toluene to the system under nitrogen protection, and stir the resulting system at room temperature for 0.5h to fully remove residual water in the system. The reaction system was then cooled to -40°C and stirred, and a certain amount of TMSOTf was added. Stirring was maintained at this temperature until TLC showed that the reaction was complete (about 3h). Triethylamine was added to the system to quench the reaction, filter, concentrate, wet-load, and separate by silica gel column chromatography to obtain compound 2a (white solid, 60% yield). 1 H NMR (400 MHz, chloroform-d) δ7.72 (d, J = 6.4 Hz, 2H), 7.64 (d, J = 6.4 Hz, 2H), 7.49–7.47 (m, 2H), 7.44–7.23 (m, 21H), 7.04 (d, J = 7.8 Hz, 2H), 5.57 (d, J = 7.2 Hz, 1H), 5.17 (d, J = 7.2 Hz, 1H), 5.10 (dd, J = 9.7, 8.1 Hz, 1H), 4.93 (d, J = 8.0 Hz, 1H), 4.82 (d, J = 11.4 Hz, 1H),4.74(d,J=10.9Hz,1H),4.66–4.60(m,3H),4.20(dd,J=9.2,9.2Hz,1H),4.05–4.02(m,2H),3.97(dd,J=9.3,9.2Hz,1H ),3.92–3.76(m,3H),3.74–3.67(m,1H),3.60(s,3H),3.51(dd,J=9.2,9.2Hz,1H),2.32(s,3H),1.82(s,3H),1.07(s,9H).C 59 H 66 N3O 11 SSi + [M+H] + MS (ESI) m / z, calculated value 1052.4, found value 1052.7.

[0438] Step B: Synthesis of compound 2b

[0439] Compound 2a (1 equivalent), thioglycolic acid (CAS: 68-11-1) / pyridine / chloroform (v / v / v = 1:1:1) were added to a reaction flask. The system was heated to 60°C and stirred. The reaction progress was monitored by TLC. After completion of the reaction (approximately 12 h), most of the solvent was removed by concentration under reduced pressure. An appropriate amount of ethyl acetate was added, and the mixture was washed with saturated sodium bicarbonate solution. After extraction and separation, the organic phase was washed with 1M hydrochloric acid solution and then washed with saturated sodium bicarbonate solution. The resulting organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 2b (white solid, 71% yield). 1 H NMR (400MHz, chloroform-d) δ7.70(d,J=6.8Hz,2H),7.66(d,J=7.2Hz,2H),7.44–7.28(m,20H),7.26–7.21(m,3H),7.00(d,J=7.9Hz,2H),5. 68(d,J=4.8Hz,1H),5.36–5.30(m,1H),5.09(dd,J=9.5,8.1Hz,1H),4.92(d,J=12.4Hz,1H),4.85(d,J=11.5Hz,1H),4.75–4.66(m,3 H),4.63–4.57(m,2H),4.29(ddd,J=9.3,7.7,4.8Hz,1H),4.10(t,J=7.8Hz,1H),4.01(dd,J=11.5,3.2Hz,1H),3.94–3.89(m,2H),3. 86–3.84(m,1H),3.80(dd,J=11.5,2.5Hz,1H),3.67(s,3H),3.60–3.51(m,2H),2.28(s,3H),1.85(s,3H),1.82(s,3H),1.07(s,9H). C 61 H 70 NO 12 SSi + [M+H] + MS (ESI) m / z, calculated value 1068.4, found value 1068.4.

[0440] Step C: Synthesis of compound 2c

[0441] Compound 2b (1 equivalent) and tetrahydrofuran were added to the reaction flask, and then TBAF (2 equivalents, 1M in THF) was added to the system. The mixture was stirred at room temperature and the reaction progress was monitored by TLC. After the reaction was complete (about 10 h), saturated ammonium chloride solution was added to the system to quench the reaction. The mixture was then extracted with ethyl acetate. The combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and wet loaded. Compound 2c was initially purified by silica gel column chromatography to obtain compound 2c (white solid, 56% crude yield). 45 H 52 NO 12 S + [M+H] + MS (ESI) m / z, calculated value 830.3, found value 830.6.

[0442] Step D: Synthesis of compound 2d

[0443] Compound 2c was dissolved in THF / MeOH (v / v = 3:1), and 1 M aqueous sodium hydroxide solution was slowly added to adjust the system to pH = 12. The resulting reaction solution was stirred at room temperature and the reaction progress was monitored by HPLC. After the reaction was sufficient (about 12 h), 1 M hydrochloric acid was added to the system to adjust the pH to 7-8. The solution was concentrated under reduced pressure to remove most of the organic solvent. The resulting crude product was purified by HPLC to obtain compound 2d (white solid, 82% yield). 1 H NMR (400 MHz, chloroform-d) δ7.43–7.27 (m, 17H), 7.07 (d, J = 7.9 Hz, 2H), 5.54 (d, J = 5.0 Hz, 1H), 5.33 (d, J = 8.0 Hz, 1H), 4.93 (d, J = 12.1 Hz, 1H), 4.86 (d, J = 11.3 Hz, 1H), 4.82–4.77 (m, 3H), 4.74 (d, J = 6.8 Hz, 1H), 4.69 (d,J=12.2Hz,1H),4.36(ddd,J=10.6,7.9,5.0Hz,1H),4.25–4.22(m,1H),4.13–4.02(m,3H),3.88(t, J=8.6Hz,1H),3.81(dd,J=12.7,2.2Hz,1H),3.74(dd,J=10.7,8.5Hz,1H),3.68–3.59(m,2H),3.40(br s,1H),2.30(s,3H),1.77(s,3H). 13C NMR (100 MHz, chloroform-d) δ 170.73, 169.33, 138.20, 138.06, 137.81, 137.55, 132.37, 129.99, 129.44, 128.84, 128.76, 128.58, 128.42, 128.30, 127.90, 103.24, 88.40,83.43,78.89,77.85,77.35,77.24,77.03,76.92,76.72,75.17,74.93,74.57,74.02,73.93,72.95,60.66,52.95,23.08,21.06 (three carbon signals in the aromatic region overlap and do not appear). C 42 H 46 NO 11 S - [MH] - MS (ESI) m / z, calculated value 772.3, found value 772.2.

[0444] Step E: Synthesis of compound 2e

[0445] Weigh compound 2d and dissolve it in acetone. Stir in an ice-water bath for 5 minutes to cool. Weigh NBS and add it to the reaction mixture. Incubate in an ice-water bath for approximately 1 hour. Take a sample and send it to HPLC to confirm the completion of the reaction. After quenching with a saturated sodium thiosulfate solution, concentrate to remove the acetone, prepare by prep-HPLC, and lyophilize to obtain 2e (white solid, 45% yield). 35 H 40 NO 12 - [MH] - MS (ESI) m / z, calculated value 666.3, found value 666.4.

[0446] Step F: Synthesis of compound 2

[0447] At room temperature, compound 2e, tetrahydrofuran, methanol, and palladium-carbon catalyst were added sequentially to a 50 mL single-necked flask. The reaction system was stirred under a hydrogen atmosphere until all the starting material disappeared as determined by TLC. The mixture was filtered, concentrated under reduced pressure, and pumped dry to obtain compound 2 (white solid, 95% yield). 14 H 22 NO 12 - [MH + ] - MS (ESI) m / z, calculated value 396.1, found value 396.1.

[0448] Preparation of disaccharide substrate compound 3

[0449] The following steps were used to prepare compound 3, the structure of which is shown below:

[0450] The preparation process of compound 3 refers to the synthesis method of compound 2, and the specific route is as follows:

[0451] The final product, compound 3, was confirmed by mass spectrometry. 14 H 22 NO 12 - [MH + ] - MS (ESI) m / z, calculated value 396.1, found value 396.0.

[0452] Preparation of disaccharide substrate compound 4

[0453] The following steps were used to prepare compound 4, whose structure is as follows:

[0454] The preparation process of compound 4 is similar to the synthesis method of compound 2, and the specific route is as follows:

[0455] The final product, compound 4, was confirmed by mass spectrometry. 14 H 22 NO 12 - [MH + ] - MS (ESI) m / z, calculated value 396.1, found value 396.1.

[0456] Preparation of disaccharide substrate compound 5

[0457] The following steps were used to prepare compound 5, the structure of which is shown below:

[0458] The preparation process of compound 5 refers to the synthesis method of compound 2, and the specific route is as follows:

[0459] The final product, compound 5, was confirmed by mass spectrometry. 14 H 22 NO 12 - [MH + ] - MS (ESI) m / z, calculated value 396.1, found value 396.0.

[0460] Example 3.2 Preparation of linker-payload (LP)

[0461] Preparation of LP-1

[0462] The structure of linker payload 1 (LP-1) is as follows:

[0463] (1) Preparation of compound LP-1b

[0464] At room temperature, compound 1 (0.5-5.0 equivalents), compound LP-1a (1.0 equivalent, GGG-VC-PAB-MMAE, CAS No. 2684216-48-4, commercially available), DMF, DIPEA (1-10 equivalents), and HATU (0.5-10 equivalents) were added sequentially to a 10 mL single-necked vial. The resulting reaction solution was stirred at room temperature until the reaction was complete as monitored by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-1b (white solid, yield 81.2%). 78 H 126 N 14 O 26 2+ [M+2H] 2+ MS (ESI) m / z, calculated value 837.4, found value 837.9.

[0465] (2) Preparation of compound LP-1

[0466] At room temperature, compound LP-1b (21.7 mg, 0.013 mmol, 1 equivalent), H2O, Et3N (1–100 equivalents), and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1–100 equivalents) were added sequentially to a 10 mL single-necked vial. The resulting reaction solution was stirred at room temperature and monitored by HPLC until the reaction was complete. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-1 (15.3 mg, 71.3% yield, as a white solid). 78 H 124 N 14 O 25 2+ [M+2H] 2+ MS (ESI) m / z, calculated value 828.4, found value 828.6.

[0467] Preparation of LP-2

[0468] The structure of linker payload 2 (LP-2) is as follows:

[0469] The preparation process is as follows:

[0470] 20.1 Step A: Preparation of Compound LP-2a

[0471] At room temperature, compound 2 (0.5-5.0 equivalents), compound LP-1a (1.0 equivalent, GGG-VC-PAB-MMAE, CAS No. 2684216-48-4, commercially available), DMF, DIPEA (1-10 equivalents), and HATU (0.5-10 equivalents) were added sequentially to a 10 mL single-necked vial. The resulting reaction solution was stirred at room temperature until the reaction was complete as monitored by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-2a (white solid, 84% yield). 78 H 126 N 14 O 26 2+ [M+2H] 2+ MS (ESI) m / z, calculated value 837.4, found value 837.8.

[0472] 20.2 Step B: Preparation of Compound LP-2

[0473] At room temperature, compound LP-2a (1 equivalent), H2O, Et3N (1–100 equivalents), and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1–100 equivalents) were added sequentially to a 10 mL single-necked vial. The resulting reaction solution was stirred at 0°C and monitored by HPLC until the reaction was complete. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-2 (yield 87%, white solid). 78 H 124 N 14 O 25 2+ [M+2H] 2+ MS (ESI) m / z, calculated value 828.4, found value 828.7.

[0474] Preparation of LP-3, LP-4, and LP-5

[0475] Using similar steps to prepare LP-2, the linker payloads LP-3, LP-4, and LP-5 with the following structures were prepared.

[0476] Preparation of LP-6

[0477] (1) Synthesis of LP-6-1

[0478] Step A: Synthesis of intermediate LP-6-1b

[0479] Compound LP-6-1a (1.0 equivalent) and DMF (dissolved at 1 g / mL) were added to a reaction flask and stirred to dissolve under a nitrogen atmosphere. After cooling to 0-5°C, DIEA (3 equivalents) was added dropwise. The resulting system was then stirred at 5°C for 10 minutes, followed by benzyl bromide (1.3 equivalents). After the addition was complete, the reaction system was allowed to naturally warm to room temperature and stirred for 16 hours. The reaction solution was slowly poured into ice water, and methyl tert-butyl ether was added, stirred, and allowed to stand for separation. The aqueous phase was extracted four times with methyl tert-butyl ether. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude yellow oil. The product was wet-loaded and purified by silica gel column chromatography (eluent: PE / EA = 6:1) to obtain product LP-6-1b (pale yellow oil, quantitative yield).

[0480] Step B: Synthesis of intermediate LP-6-1d

[0481] Under nitrogen, intermediate LP-6-1b (2.0 equivalents), compound LP-6-1c (1.0 equivalents), and THF (dissolved at 10 g / mL) were added to a reaction flask and stirred to dissolve. TsOH (0.1 equivalents) was added to the reaction, and the system was allowed to react at room temperature for 4 hours. The reaction solution was slowly poured into ice water and extracted three times with ethyl acetate. The combined organic phases were washed sequentially with saturated sodium bicarbonate aqueous solution, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified by silica gel column chromatography (eluent: PE / EA = 1:1) to obtain the product LP-6-1d (white solid, 40% yield).

[0482] Step C: Synthesis of intermediate LP-6-1e

[0483] Under nitrogen protection, compound LP-6-1d and N,N-dimethylacetamide (DMAc, dissolution concentration of 10 g / mL) were added to the reaction flask and stirred to dissolve. The system was cooled to 14-18°C, and DBU (0.5 equivalent) was added dropwise. The reaction was maintained at this temperature until TLC showed that the reaction was complete (about 1.5 h) to obtain the intermediate LP-6-1e, which was directly used in the next reaction without purification.

[0484] Step D: Synthesis of intermediate LP-6-1g

[0485] The reaction mixture from the previous step was cooled to 0-5°C, and 4-methylbenzenesulfonate pyridinium (PPTS, 0.5 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 1.0 equivalent), 1-hydroxybenzotriazole (HOBT, 1.0 equivalent), and LP-6-1f (0.85 equivalent) were added in sequence. The reaction system was allowed to react at 0-10°C until LCMS indicated completion (approximately 4 hours). The reaction mixture was added to ice water and extracted once with 2-methyltetrahydrofuran. The aqueous phase was then extracted twice with 2-methyltetrahydrofuran. The organic phases were combined and washed sequentially with 0.5M hydrochloric acid, saturated aqueous NaHCO3, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: DCM / MeOH) to obtain the product LP-6-1g (white solid, yield 78%).

[0486] Step E: Synthesis of intermediate LP-6-1h

[0487] Under nitrogen, add LP-6-1g and DMAc (dissolved at 10g / mL) to a reaction flask and stir to dissolve. Cool to 14-18°C, then add DBU (0.5 equivalents) dropwise. Stir at this temperature for 1.5 hours. Monitor the reaction progress by TLC. Upon completion, the intermediate LP-6-1h is obtained and used directly in the next step without purification.

[0488] Step F: Synthesis of intermediate LP-6-1j

[0489] The reaction solution of LP-6-1h in the previous step was cooled to 0-5°C, and PPTS (0.5 equivalent), EDCI (1 equivalent), HOBT (1 equivalent) and compound 3i (0.85 equivalent) were added. The reaction was carried out at 0-10°C for 3-4 hours. The reaction progress was monitored by LCMS. After the reaction was complete, the reaction solution was added to ice water and extracted once with 2-methyltetrahydrofuran. The aqueous phase was extracted twice with 2-methyltetrahydrofuran. The organic phases were combined and washed with 0.5M hydrochloric acid, saturated NaHCO3 aqueous solution, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, dry-mixed, and purified by column chromatography (eluent: DCM / MeOH) to obtain LP-6-1j (white solid, yield 50%).

[0490] Step G: Synthesis of compound LP-6-1

[0491] Under nitrogen, intermediate LP-6-1j was dissolved in DCM (15 g / mL) and DBU (0.5 equivalent) was added dropwise at 20°C. The reaction was stirred and maintained at this temperature until HPLC indicated completion. DCM was then added to dilute the reaction solution, and the sample was directly wet-loaded and purified by column chromatography (eluent: DCM / MeOH) to obtain compound LP-6-1 (white solid, 82% yield). 34 H 47 N6O 10 + [M+H] + MS (ESI) m / z, calculated value 699.4, found value 699.6.

[0492] (2) Synthesis of LP-6-2

[0493] LP-6-2 was synthesized using peptide solid phase synthesis method. The steps are as follows:

[0494] Step 1: Preparation of NH2-Asp(OtBu)-Rink amide resin

[0495] Weigh 400g of Rink amide resin into a reactor and soak it in 2400mL of DCM for 0.5h to fully swell the resin. Drain and drain. Add 2400mL of decapping reagent and wash. Drain and drain. Add 2400mL of decapping reagent and stir under nitrogen at 25±1°C for 0.5h. Drain and drain. Wash twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF. Drain and drain. Kaiser detector indicates blue.

[0496] Weigh 88.87g of Fmoc-Asp(OtBu)-OH and 29.19g of HOBT and dissolve them in 2000mL of DMF and 80mL of DIC. Place in a -10°C ice bath for 0.5h, then slowly add the mixture to the reactor. Stir under nitrogen purge for 2h at room temperature and drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, drain. A Kaiser assay indicates a light blue color. Add 2400mL of DCM and 60mL of capping reagent, stir under nitrogen purge at 25±1°C for 1h, and drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, drain. A Kaiser assay indicates a colorless color.

[0497] Add 2400 mL of decapping reagent to wash and drain. Add 2400 mL of decapping reagent and stir under nitrogen at 25±1°C for 0.5 h, then drain. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, drain. Kaiser assay results in blue.

[0498] Step 2: Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin

[0499] Weigh 131.64g of Fmoc-PEG4-OH and 48.64g of HOBT and dissolve them in 2000mL of DMF and 80.0ml of DIC. Place in a -10°C ice bath for 0.5h, then slowly add the mixture to the reactor. Stir under nitrogen purge at room temperature for 2-4h, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, then drain. Kaiser assay indicates colorless.

[0500] Add 2400 mL of decapping reagent to wash and drain. Add another 2400 mL of decapping reagent and stir under nitrogen at 25±1°C for 0.5 h. Drain. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, drain. Kaiser assay indicates blue.

[0501] Step 3: Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0502] Weigh 222.18g of Fmoc-Asp(OtBu)-OH and 72.96g of HOBT and dissolve them in 2000mL of DMF and 80mL of DIC. Place in a -10°C ice bath for 0.5h, then slowly add the mixture to the reactor. Stir and react under nitrogen bubble for 2-4h at room temperature, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, then drain. Kaiser assay indicates a colorless solution.

[0503] Add 2400 mL of decapping reagent, wash, and drain. Add another 2400 mL of decapping reagent, stir and react at 25 ± 1°C under nitrogen for 0.5 h, and drain. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, drain. Kaiser assay indicates blue.

[0504] Step 4: Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0505] Weigh 191.75g ​​of Dde-Lys(Fmoc)-OH and 48.64g of HOBT and dissolve them in 2000mL of DMF and 80.0ml of DIC. Place in a -10°C ice bath for 0.5h, then slowly add the mixture to the reactor. Stir and react under nitrogen bubbling at room temperature for 2-4h, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, then drain. Kaiser assay indicates colorless.

[0506] Add 2400 ± 100 mL of decapping reagent to wash and drain. Add another 2400 mL of decapping reagent and stir under nitrogen at 25 ± 1°C for 0.5 h. Drain. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, drain. Kaiser assay indicates blue.

[0507] Step 5: Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0508] Weigh 170.84g of m-PEG12-CH2CH2COOH and 48.64g of HOBT and dissolve them in 2000mL of DMF and 80.0ml of DIC. Place in a -10°C ice bath for 0.5h, then slowly add the mixture to the reactor. Stir under nitrogen at room temperature for 2-4h, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, then drain. Kaiser assay indicates colorless.

[0509] Step 6: Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0510] Add 2400 mL of de-DDE reagent and stir at 25±1°C for 10 min under nitrogen bubble. Drain and repeat this operation three times. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM and 2400 mL of DMF respectively. Drain and the Kaiser detector shows blue.

[0511] Step 7: Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0512] Weigh 111.08g of Fmoc-Gly-Gly-Gly-OH and 48.64g of HOBT and dissolve them in 2000mL of DMF solution and 80.0ml of DIC. Place in a -10℃ ice bath for 0.5h, then slowly add them to the reactor for reaction. Stir and react under nitrogen bubbling at room temperature for 2-4h, then drain. Wash with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF twice each, drain, and Kaiser test. The resin peptide is colorless. Wash three times with 2400mL of anhydrous ethanol, drain, and wait for cleavage.

[0513] Step 8: Preparation of LP-6-2

[0514] Add 10,000 mL of cleavage reagent (TFA:TIS:H2O = 95:2.5:2.5) to a 10 L reactor and cool to -10 ± 2°C. Add the dried, weighed resin and heat to 20 ± 5°C, stirring under nitrogen for 2 h. Filter and wash the resin once with 100 mL of TFA. Combine the filtrate and washings.

[0515] Add 40 L of pre-cooled (below -10°C) cold ether, stir for 10 minutes and then centrifuge to precipitate. After centrifugation, discard the supernatant, mix the precipitate with cold ether and shake it evenly, then centrifuge again (repeat this step 3 times, each time using 10 L, 10 L, and 10 L. Set the centrifugal speed to 3600 rpm, the centrifugation time to 5 minutes, and the temperature of the centrifuge cavity to -5°C).

[0516] The precipitate was collected to obtain the crude product of LP-6-2, which was purified by Prep-HPLC and lyophilized to obtain LP-6-2. 74 H 121 N9O 31 2+ [M+2H] 2+ MS (ESI) m / z, calculated value 815.9, found value 816.3.

[0517] (3) Synthesis of LP-6-3

[0518] Step A: Synthesis of intermediate LP-6-3a

[0519] Compound LP-6-1 (2.2 equivalents) and compound LP-6-2 (1.0 equivalents) were added to the reaction flask and dissolved in DMF. DIPEA (5.0 equivalents) was then added and stirred evenly. HATU (2.5 equivalents) was added to the system and the reaction was allowed to proceed at room temperature. HPLC monitoring was performed until the reaction was complete (approximately 2 hours). The reaction system was directly prepared by prep-HPLC, and the prepared solution was lyophilized to obtain compound LP-6-3a (white solid, yield 52%). 143 H 212 O 48 N 21 3+ [M+3H] 3+ The MS (ESI) m / z was calculated to be 997.2 and found to be 875.9 (some functional groups would be detached after ionization).

[0520] Step B: Synthesis of intermediate LP-6-3b

[0521] Compound LP-6-3a was dissolved in purified water, and a certain amount of palladium hydroxide (10 wt% Pd(OH)2 on carbon) was added. The system was replaced with hydrogen three times and stirred at room temperature for 1.5 h. During this period, the reaction progress was monitored and the reaction was stopped immediately after the starting material disappeared to prevent the increase of de-Fmoc products. The reaction solution was filtered and prepared by prep-HPLC to obtain compound LP-6-3b (white solid, yield 76%). 128 H 200 O 48 N 21 3+ [M+3H] 3+ The MS (ESI) m / z was calculated to be 937.1 and the actual value was 875.9 (some functional groups would be detached after ionization).

[0522] Step C: Synthesis of intermediate LP-6-3d

[0523] Compound LP-6-3b (1.0 equivalent) and compound LP-6-3c (2.2 equivalents) were dissolved in DMF, DIPEA (5.0 equivalents) was added and stirred uniformly, followed by addition of HATU (2.5 equivalents). The reaction was allowed to proceed at room temperature and monitored by HPLC until the reaction was complete (approximately 16 h). The reaction system was directly prepared by prep-HPLC, and the prepared solution was lyophilized to obtain compound LP-6-3d (yellow solid, yield 66%). 174 H 232 O 55 Cl2F2N 27 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1229.2, found value 1229.3.

[0524] Step D: Synthesis of compound LP-6-3

[0525] Compound LP-6-3d was dissolved in DMF, diethylamine was added, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until the reaction was complete (about 0.5 h). After the reaction was completed, the pH was adjusted to neutral, and then prep-HPLC was used for preparation. After freeze-drying, compound LP-6-3 (yellow solid, yield 73%) was obtained. 159 H 222 O 53 Cl2F2N 27 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1155.2, found value 1155.3.

[0526] For the synthesis of compound LP-6-3c, please refer to patent CN202211428194.6.

[0527] (4) Synthesis of LP-6

[0528] Synthesis of LP-6a

[0529] Compound 1 (0.5-5.0 equivalents) and compound LP-6-3 (1.0 equivalents) were dissolved in DMF, DIPEA (1-10 equivalents) was added and stirred at 0°C. HATU (0.5-10 equivalents) was added and the reaction system was stirred at 0°C. The reaction progress was monitored by HPLC until the reaction was complete (approximately 2 hours). The reaction system was directly prepared by prep-HPLC, and the preparative solution was lyophilized to obtain compound LP-6a (yellow solid, 63% yield). 173 H 243 O 64 Cl2F2N 28 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1281.5, found value 1281.8.

[0530] Synthesis of LP-6

[0531] Compound LP-6a was weighed and dissolved in purified water. After stirring and cooling in an ice-water bath, Et3N (1-100 equivalents) and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1-100 equivalents) were added. The reaction progress was monitored by HPLC. After the reaction was complete, it was purified by prep-HPLC. The prepared solution was lyophilized to obtain compound LP-6 (light yellow solid, yield 74%). 173 H 241 O63 Cl2F2N 28 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1275.5, found value 1275.9.

[0532] Preparation of LP-7

[0533] The structure of LP-7 is as follows:

[0534] The synthetic route is as follows:

[0535] Step A: Synthesis of compound LP-7b

[0536] Compound LP-7a was synthesized by a peptide solid phase synthesis method, referring to a similar method to LP-6-2.

[0537] Compound LP-7a (1.0 equivalent) and compound LP-6-1 (3.6 equivalents) were added to a reaction flask and dissolved in DMF. DIPEA (6.0 equivalents) was then added and stirred evenly. HATU (3.6 equivalents) was added to the system and the reaction was allowed to proceed at room temperature. HPLC monitoring was performed until the reaction was complete (approximately 2 h). The reaction system was directly prepared by prep-HPLC. The preparative solution was lyophilized to obtain compound LP-7b (white solid, yield 56%). 191 H 280 O 66 N 29 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1345.3, found value 1345.8.

[0538] Step B: Synthesis of intermediate LP-7c

[0539] Compound LP-7b was dissolved in purified water, and a certain amount of palladium hydroxide (10 wt% Pd(OH)2 on activated carbon) was added. The system was replaced with hydrogen three times and stirred at room temperature for 1.5 h. During this period, the reaction progress was monitored. The reaction solution was filtered and directly prepared by prep-HPLC to obtain compound LP-7c (white solid, yield 80%). 170 H 262 O 66 N 29 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1255.3, found value 1255.9.

[0540] Step C: Synthesis of intermediate LP-7d

[0541] Compound LP-7c (1.0 equivalent) and compound LP-6-3 (1-10 equivalents) were dissolved in DMF, and DIPEA (1-20 equivalents) was added and stirred evenly. HATU (1-10 equivalents) was then added and reacted at room temperature. The reaction was monitored by HPLC until the reaction was complete (about 2 h). The reaction was directly used for the next step. 239 H 313 O 75 Cl3F3N 38 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1692.4, found value 1692.9.

[0542] Step D: Synthesis of compound LP-7e

[0543] Compound LP-7d was dissolved in DMF, diethylamine was added, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until the reaction was complete (about 0.5 h). After the reaction was completed, the pH was adjusted to neutral, and then prep-HPLC was used for preparation. After freeze-drying, compound LP-7e (yellow solid, yield 73%) was obtained. 224 H 303 O 73 Cl3F3N 38 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1618.3, found value 1618.5.

[0544] Step E: Synthesis of compound LP-7f

[0545] Compound 1 (0.5-5.0 equivalents) and compound LP-7e (1.0 equivalent) were dissolved in DMF, DIPEA (1-10 equivalents) were added, and the mixture was stirred at 0°C. HATU (0.5-10 equivalents) was added, and the reaction system was stirred at 0°C. The reaction progress was monitored by HPLC until the reaction was complete (approximately 2 hours). The reaction system was directly prepared by prep-HPLC, and the preparative solution was lyophilized to obtain compound LP-7f (yellow solid, 65% yield). 238 H 325 O 84 Cl3F3N 39 4+ [M+4H] 4+ MS (ESI) m / z, calculated value 1308.8, found value 1309.0.

[0546] 15.6Step F: Synthesis of Compound LP-7

[0547] Compound LP-7f was weighed and dissolved in purified water. After stirring and cooling in an ice-water bath, Et3N (1-100 equivalents) and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1-100 equivalents) were added. The reaction progress was monitored by HPLC. After the reaction was complete, it was purified by prep-HPLC. The prepared solution was lyophilized to obtain compound LP-7 (light yellow solid, 50% yield).

[0548] Example 4. Preparation of anti-Her2 antibodies

[0549] Anti-human ErbB2 / Her2 antibody T-LCCT L For the production, purification and identification of -HC (mAb-1) and anti-human TROP2 antibody mAb-2, please refer to Example 1 of patent CN106856656B, the entire text of CN106856656B is incorporated herein by reference.

[0550] Example 5. Suspension coupling

[0551] Solid phase preparation of ADC-1

[0552] ADC drugs are prepared based on the coupling reaction of Halo-Endo S2-His catalyzing the antibody and the linker-payload. In the endonuclease buffer, the antibody and the linker-payload are thoroughly mixed at an appropriate molar ratio (1:1 to 1:100), and the Halo-Endo S2-His medium is added and mixed. The coupling reaction in the mixed state is carried out at 4-40°C for 0.5-20 hours. After the reaction is completed, centrifugation is performed, and the solid phase coupling reaction mixture is removed and purified, ultrafiltered or dialyzed to remove unreacted drug intermediates. The purified ADC1 is stored in 1×PBS pH7.4 and stored at 4°C or -80°C.

[0553] 1) SDS-PAGE analysis

[0554] After the conjugation reaction, the purity and conjugation efficiency of ADC-1 were determined by SDS-PAGE. The SDS-PAGE results for ADC-1 are shown in Figure 3. The conjugation reaction is localized to the antibody's heavy chain (a significant molecular weight shift is observed between the conjugated cytotoxin heavy chain and the unconjugated heavy chain). No unconjugated cytotoxin heavy chain was detected in the conjugated product, demonstrating a conjugation efficiency exceeding 95%.

[0555] 2) HIC-HPLC analysis

[0556] HIC-HPLC: A Proteomix HIC Butyl-NP5 4.6*100mm 5μm Non-Porous column was used; the column temperature was 30°C; the mobile phase A was 1.5M ammonium sulfate + 20mM phosphate buffer, pH 7.0, and the mobile phase B was 20mM phosphate buffer: isopropanol = 7:3 (v / v); the flow rate was 0.8mL / min; the gradient method was from 10% to 100% phase B within 8 minutes; and the detection wavelength was 280nm to detect the DAR distribution of the ADC1 drug.

[0557] The test results are shown in FIG4 and Table 2. The amount of antibodies not coupled to compound LP-1 was less than 1%. The coupled product was mainly DAR2, and the DAR value of the ADC-1 drug was 1.93.

[0558] Table 2. Halo-Endo S2-His coupling efficiency

[0559] The coupling efficiency in Table 2 is 1.93 / 2=96.5%

[0560] 3) Mass spectrometry

[0561] High-precision molecular weight mass spectrometry (ESI-MS) analysis of ADC-1

[0562] ADC-1 was analyzed and detected using high-precision molecular weight mass spectrometry. The results showed that its apparent molecular weight was 150539.27 and the theoretical molecular weight was 150538.78, which was in line with expectations and confirmed that each heavy chain Fc end was coupled to a cytotoxin molecule.

[0563] Example 6. Column coupling

[0564] Separately prepare antibody and small molecule toxin (LP-1) reaction solutions at concentrations of 1-100 mg / ml for the antibody and 0.1-50 mg / ml for the small molecule. Load the treated immobilized enzyme (Halo-Endo S2-His) medium onto a column and heat to 10-40°C via air or water bath conduction, preheating for >30 minutes. Mix the antibody and small molecule reaction solutions in a fixed ratio and allow the reaction to proceed via a coupling column. The effluent is the ADC drug, with a retention time on the coupling column ranging from 5 minutes to 24 hours. Samples of the coupling reaction solution are analyzed by SDS-PAGE, HIC-HPLC, and RP-HPLC.

[0565] 1) SDS-PAGE analysis

[0566] After the conjugation reaction, the purity and conjugation efficiency of ADC-1 were determined by SDS-PAGE. SDS-PAGE analysis of ADC-1 demonstrated that the conjugation reaction occurred specifically on the heavy chain of the antibody, with no unconjugated cytotoxin heavy chain detected in the conjugated product, demonstrating a conjugation efficiency exceeding 95%.

[0567] 2) HIC-HPLC analysis

[0568] The test results showed that the DAR of ADC-1 after the coupling reaction was 1.93 and the coupling efficiency was 96.5%.

[0569] The efficiency of both column coupling and suspension coupling can reach over 95%. The results of column coupling are used to support the linear production scale-up of ADC drugs.

[0570] The specific operation process of column coupling is as follows: install a suitable pipeline (such as peristaltic pump silicone tube) to a suitable pump head (such as peristaltic pump head (Chongqing Jieheng, BT-600CA / DG4(10))) and balance the pipeline. Then connect the outlet end of the pipeline to the inlet end of the coupling column, and use another section of pipeline to connect to the lower end of the coupling column. Place the connected coupling column in a temperature-controlled device (e.g., a hybridization oven (UVP / HB-1000 Hybridizer)) set at 10-40°C. Place the outlet of the tubing at the lower end of the coupling column outside the temperature-controlled device. Place the inlet end of the tubing in the coupling reaction solution, start the pump speed to conduct the coupling reaction, and collect the coupling reaction solution. After the coupling reaction solution is loaded, rinse the coupling column with coupling equilibration buffer and collect the same collection volume; the effluent is the ADC drug. After collection, the concentration of the collected solution is measured using a UV spectrophotometer and purified, ultrafiltered, or dialyzed to remove unreacted drug intermediates. The purified ADC is stored in 1× PBS pH 7.4 at 4°C or -80°C. Samples of the coupling reaction solution are analyzed by SDS-PAGE, HIC-HPLC, or RP-HPLC.

[0571] Solid-phase coupling column reaction can achieve linear amplification. Under the same conditions of key process parameters (such as retention time), the corresponding process (such as pump flow rate) is adjusted according to the size of the coupling column to achieve linear amplification of the coupling column to several hundred milliliters (500 mL), several liters (3 L) or larger volumes of coupling columns. Glycosidase coupling column catalysis is achieved on a laboratory scale, and DAR detection is performed offline; a larger coupling column is used to achieve linear amplification of the coupled drug at a higher flow rate, as well as online DAR value detection. The coupling device in patent application WO2022170676A is suitable for the glycosylation process of the present invention. This example verifies the feasibility of applying coupling equipment of different scales to the glycosylation coupling platform.

[0572] Example 7. Separation of Halo-Endo S2-His and ADC

[0573] When the pH value of the environment is above its isoelectric point, the protein will bind to positively charged packings, namely anion exchangers; when the pH value of the environment is below its isoelectric point, the protein will bind to negatively charged packings, namely cation exchangers. The isoelectric point of Halo-Endo S2-His is 5.5. At pH 6.0-8.0, the surface of Halo-Endo S2-His protein has a negative charge. The Halo-Endo S2-His protein will bind to negatively charged packings, namely cation exchangers, in flow-through mode; the Halo-Endo S2-His protein will bind to positively charged packings, namely anion exchangers. The isoelectric point of ADC is typically 8-9. At pH 6.0-8.0, the surface of ADC protein has a positive charge. The ADC will bind to negatively charged packings, namely cation exchangers, and the ADC will bind to positively charged packings, namely anion exchangers, in flow-through mode. Halo-Endo S2-His and ADC have a significant difference in isoelectric point (IP) greater than 2. Selecting a pH 6.0-8.0 buffer allows for effective separation of ADC and Halo-Endo S2-His via ion exchange chromatography. This means that Halo-Endo S2-His is used to specifically catalyze the ADC. Even if a small amount of Halo-Endo S2-His protein immobilized on the column is lost in the ADC product, subsequent anion and cation chromatography can effectively remove the Halo-Endo S2-His.

[0574] 1) Anion chromatography (AEX)

[0575] The column was packed with Q Sepharose FF and equilibrated with 20mM Tris-HCl, pH 7.5, before loading the sample. After loading, the column was equilibrated to baseline with 20mM Tris-HCl, pH 7.5. Finally, the sample was eluted with 20mM Tris-HCl, 1M NaCl, pH 7.5, and cleaned with 1M NaOH for CIP (cleaning in place of solvent). ADC and Halo-Endo S2-His were purified using the same method and equilibrated with the same equilibrated sorbent. ADC-1 and Halo-Endo S2-His were also purified after mixing.

[0576] The results are shown in FIG5A : ADC-1 sample was passed through the Q FF column in flow-through mode at pH 7.5.

[0577] The Halo-Endo S2-His sample was passed through a QFF column in column loading mode at pH 7.5, with 20 mM Tris-HCl, 1 M NaCl, pH 7.5, to elute the sample. As shown in Figure 5B , a mixed load of ADC-1 and Halo-Endo S2-His was effectively separated after passing through the QFF column, with ADC-1 flowing through and Halo-Endo S2-His loading remaining.

[0578] 2) Cationic ion chromatography (CEX)

[0579] The column was packed with Capto S impact cationic packing and equilibrated with 20mM citric acid / sodium citrate, pH 6.2, before loading. After loading, the column was equilibrated to baseline with 20mM citric acid / sodium citrate, pH 6.2, and the sample was eluted with 20mM citric acid / sodium citrate, 160mM NaCl, pH 6.2. The column was regenerated with 20mM citric acid / sodium citrate, 1M NaCl, pH 6.2, and then cleaned and purified with 1M NaOH. ADC and Halo-Endo S2-His were purified using the same method and packing material, as well as a mixture of ADC and Halo-Endo S2-His.

[0580] The results are shown in FIG6A : ADC-1 sample was passed through a Capto S impact cationic chromatography column in column mode at pH 6.2.

[0581] The Halo-Endo S2-His sample was run through a Capto S impact cationic chromatography column at pH 7.5 in flow-through mode. As shown in Figure 6B , a mixed sample of ADC-1 and Halo-Endo S2-His was effectively separated after passing through a Capto S impact cationic chromatography column, with ADC-1 attached to the column and Halo-Endo S2-His flowing through.

[0582] Example 8 Detection of affinity of ADC-1 for ErbB2 / Her2 on cell surface

[0583] Take Her2 ECD and use CBS coating solution (0.1M carbonate buffer, pH 9.6) to make 1μg / ml, 100μl / well, and coat at 25℃ for 60min. After coating, wash the plate three times with PBST and block with blocking solution (5% skim milk powder) (300μl / well) at 25℃, 200rpm, and incubate for 60min. After blocking, wash the plate three times with PBST and add antibody T-LCCT. L-HC and ADC-1 (100 μl / well) were incubated at 25°C, 200 rpm for 60 minutes. After incubation with the sample, the plate was washed three times with PBST and anti-human IgG Fc-HRP (100 μl / well) was added. The plate was incubated at 25°C, 200 rpm, and incubated for 60 minutes. After the 60-minute incubation, the color was developed with TMB (100 μl / well) for 5 minutes. The reaction was then terminated with stop solution and the OD450 reading was read.

[0584] Elisa test results showed T-LCCT L There was no significant difference in the antigen affinity of -HC naked mAb and ADC-1 for Her2 ECD, as shown in Figure 7 .

[0585] Example 9. Effect of ADC-1 on the proliferation of tumor cells with different ErbB2 / Her2 expression levels

[0586] 1) ErbB2 / Her2-positive human breast cancer cells BT-474, ErbB2 / Her2-positive human gastric cancer cells NCI-N87, and ErbB2 / Her2-negative human liver cancer cells HepG2 were seeded into 96-well cell plates at 100 μl per well (containing 1,000 to 10,000 cells) and cultured overnight in a cell culture incubator at 37°C, 5% CO2, and 100% humidity.

[0587] 2) Different concentrations (10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015, 0.00051 nM) of ADC-1 or antibody T-LCCT were added to ErbB2 / Her2 positive cells cultured overnight. L -HC or different concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) of MMAE (monomethyl auristatin E); different concentrations (100, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) of ADC-1 or antibody T-LCCTL-HC or different concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) of MMAE were added to ErbB2 / Her2 negative cells cultured overnight; Puromycin (Puromycin) at a concentration of 50 μM was added to the control group. Continue incubation at 37°C for 72 to 120 hours.

[0588] 3) Remove the cell plate from the 37°C cell culture incubator and equilibrate to room temperature for approximately 30 minutes. Add 100 μl of CellTiter Glo reagent to each well, shake on a shaker for 2 minutes, and then let it rest at room temperature in the dark for 10 minutes. Measure the luminescence value (RLU) using an MD M4 microplate reader.

[0589] 4) The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 3 and Figures 8-10. Among them, ADC1 and MMAE small molecule toxins have significant inhibitory effects on ErbB2 / Her2 positive cells, and the antibody T-LCCT L -HC monoclonal antibody has a slight inhibitory effect on the proliferation of ErbB2 / Her2 positive cells, and ADC-1 is significantly better than T-LCCT L -HC monoclonal antibody and MMAE small molecule toxin. ADC-1 and T-LCCT L -HC monoclonal antibody had no inhibitory effect on ErbB2 / Her2 negative cells.

[0590] Table 3. Inhibitory effects of different drugs on tumor cell proliferation (IC 50 ,nM)

[0591] Example 10: In vivo activity test of ADC-1 (NCI-N87CDX mouse model)

[0592] The effect of ADC-1 on tumor growth in the ErbB2 / HER2 NCI-N87CDX mouse model was tested as follows:

[0593] 1) Cell culture: NCI-N87 human gastric cancer cells (ATCC, Manassas, VA, cat#CRL-5822) were collected in the logarithmic growth phase and the cell density was adjusted with Matrigel buffer (PBS:Matrigel = 1:1). 7-9 week old SPF female BALB / c nude mice were subcutaneously injected with 0.2 mL of the prepared NCI-N87 cell suspension at the right scapula. The cell inoculation size was 10 × 10 6 / Only.

[0594] 2) Measure the tumor diameter with a vernier caliper and calculate the diameter using the formula V = 0.5axb 2 Tumor volume was calculated (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor). Five days after cell inoculation, when the average tumor volume ranged from 100 to 300 mm 3Animals were randomly divided into a vehicle control group and an ADC-1 3 mg / kg group, with 5 animals in each group. Animals were dosed via tail vein injection. The control group received an equal volume of vehicle (Vehicle). The day of group administration was defined as Day 0. Tumor volumes were measured twice weekly for 35 days after administration. Tumor volumes on Day 35 were compared between groups, and T / C and TGI values ​​were calculated using tumor volumes. The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : vehicle control group RTV). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula was RTV = Vt / V0, where V0 was the average tumor volume measured during grouping, Vt was the average tumor volume at a certain measurement, and T RTV with C RTV Data were collected on the same day. Calculation of TGI (%): TGI (%) = [1 - (average tumor volume of a treatment group at the end of treatment - average tumor volume of the treatment group at the start of drug administration) / (average tumor volume of the vehicle control group at the end of treatment - average tumor volume of the vehicle control group at the start of treatment)] × 100%.

[0595] 3) Independent sample t-test was used to analyze the data between groups. All analyses were performed using SPSS 17.0. P < 0.05 was considered statistically significant.

[0596] 4) Table 4 and Figure 11 show that ADC-1 can significantly inhibit tumor growth in the NCI-N87CDX mouse model compared with the vehicle control group.

[0597] Table 4. Inhibitory effect of ADC-1 on the NCI-N87CDX mouse model

[0598] 1. Mean ± SEM;

[0599] 2. T / C% = T RTV / C RTV x 100%(T RTV :RTV in treatment group; C RTV : RTV of vehicle control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV=V 35 / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., D0), V 35 T is the average tumor volume on day 35 after administration. RTV with C RTVGet data from the same day;

[0600] 3. TGI (%) = [1-(T 35 -T0) / (V 35 -V0)]×100)

[0601] 4. P values ​​were calculated based on tumor volume.

[0602] Example 11: Preparation and Characterization of ADC-2

[0603] ADC-2 was prepared by referring to the above preparation method. The difference between ADC-2 and ADC-1 is that the linker-payload is LP-6. The characterization data of the antibody-drug conjugate ADC-2 are described below.

[0604] SDS-PAGE analysis of the antibody-drug conjugate ADC-2: After the conjugation reaction, the purity and conjugation efficiency of ADC-2 were determined by SDS-PAGE. The SDS-PAGE analysis of ADC-2 showed that the conjugation reaction occurred specifically on the heavy chain of the antibody. The ADC-2 heavy chain conjugated with the linker-payload exhibited a significant molecular weight shift compared to the heavy chain of the monoclonal antibody with the glycan removed, demonstrating successful site-specific conjugation of the linker-payload to the monoclonal antibody heavy chain. Unconjugated antibody was virtually absent in the conjugated product, with a conjugation efficiency exceeding 95%, demonstrating the expected purity of the conjugated product.

[0605] HIC-HPLC analysis of ADC-2 showed that the unconjugated cytotoxic antibody was less than 5%; the conjugated product was mainly DAR4, and the overall DAR value of ADC-2 drug was about 3.8.

[0606] Plasma stability testing of ADC-2

[0607] Experimental Design:

[0608] Mixed healthy human plasma (equal volumes of 5 males and 5 females) was collected, ADC-2 was added to a specific final concentration, and the plasma was divided into 4 portions, each containing 450 μL. The samples were incubated in a 37°C incubator. The samples were collected at 0 h, 24 h, 48 h, and 96 h, and stored in a refrigerator at -60 to -90°C after collection to detect the free load shedding rate and DAR change rate.

[0609] LC-MS detection of small molecule toxins (the small molecule toxin of LP-6 involved in this example is represented by payload)

[0610] For each sample (except Double Blank), 40 μL was added with a certain amount of internal standard precipitant (1 ng / mL DXd) and shaken for at least 10 min. For Double Blank, 40 μL of blank matrix was added with 120 μL of blank matrix without internal standard precipitant and shaken for at least 10 min. Centrifuge at 3600 g, 4°C, for 15 min, and 60 μL of supernatant was transferred to an injection vial. A certain amount of 0.1% FA ultrapure water was added and shaken for at least 3 min. Detection was performed by HPLC.

[0611] DAR detection by hybridization LC-MS

[0612] Weigh 1 g of CNBr-activated agarose (Sigma, Cat#C9142) into a 50 mL centrifuge tube. Add 50 mL of pre-chilled 1 mM HCl and incubate on a rotating mixer for at least 30 minutes at 4°C, 10 RPM. Remove the 1 mM HCl by centrifugation for a desired time. Wash the sol once with 5-10 volumes of deionized water and then three times with 0.1 M NaHCO₃. Displace the HER2 ECD using a 30 kD ultrafiltration tube (buffered with 0.1 M NaHCO₃). Mix the displaced HER2 ECD with the filler and incubate on a rotating mixer at 25°C, 10 RPM for 2 h ± 10 min for coupling. Add 5 mL of 0.1 M NaHCO₃ buffer and wash by centrifugation to remove uncoupled protein. Centrifuge for a desired time and wash three times. Add a desired amount of glycine (pH 8.0) to the filler and incubate at 2-8°C for 16 hours to block unreacted chemical groups on the filler. First, wash the filler with 5 mL of 0.1 M NaHCO₃ and centrifuge for a desired time. Then, wash the filler with a desired amount of acetate buffer and centrifuge for a desired time. Repeat this wash cycle several times. Transfer 0.1 mL of sample to a 1.5 mL EP tube and add 0.1 mL of HER2 ECD-conjugated filler. Incubate at 25°C, 10 RPM, for 2 hours. Wash three times with 1 mL of PBST and centrifuge for a desired time. After elution, centrifuge the supernatant, determine the concentration, and perform LC-MS analysis.

[0613] Result analysis:

[0614] ADC-2 incubated in healthy pooled human plasma for 0, 24, 48, and 96 hours showed DAR changes of 100.00%, 106.7%, 104.2%, and 106.5%, respectively. The shedding ratios of the small molecule toxin were only 0.006%, 0.179%, 0.373%, and 1.07%, respectively. These results demonstrate that after 96 hours of incubation at 37°C in healthy pooled human plasma, ADC-2 exhibited extremely low toxin shedding and a stable DAR. Low toxin shedding indicates a significant reduction in clinical side effects caused by free toxin. High DAR stability suggests that the antibody molecule can deliver more toxin molecules to the tumor, thereby improving drug efficacy. These two synergistic effects could significantly broaden the therapeutic window of ADC-2.

[0615] Example 13: In vitro activity test of ADC-2 (SK-BR-3, HCC1954, MDA-MB-468)

[0616] The effect of ADC-2 on the proliferation of tumor cells with different ErbB2 / HER2 expression levels was tested as follows:

[0617] Referring to the method described in Example 9, the antibody-drug conjugate ADC-2 was tested for its proliferative effects on cancer cells with varying ErbB2 / HER2 expression levels. For example, ErbB2 / HER2-positive human tumor cells, such as SK-BR-3 and HCC1954, and ErbB2 / HER2-negative human tumor cells, such as MDA-MB-468, were selected. The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 5 and Figures 13-15. ADC-2 and the small molecule toxin both showed significant anti-proliferative effects on ErbB2 / HER2-positive cells. The antibody mAb-1 showed some anti-proliferative effects on ErbB2 / HER2-positive cells, with ADC-2 significantly outperforming mAb-1. ADC-2 and mAb-1 had no inhibitory effects on ErbB2 / HER2-negative cells, demonstrating good targeting.

[0618] Table 5. Inhibitory effects of different drugs on tumor cell proliferation (IC50, nM)

[0619] Example 14: Preparation and Characterization of ADC-3

[0620] Antibody-drug conjugate ADC-3 was prepared with reference to the above preparation method. ADC-3 differs from ADC-1 in that the antibody used is the anti-TROP2 antibody mAb-2, and the linker-payload used is LP-6. Characterization data of ADC-3 are described below.

[0621] SEC-HPLC of ADC-3 showed that high molecular weight aggregates were less than 5%, and the ADC sample existed mainly in monomeric form. The damage of the coupling reaction to the antibody was negligible.

[0622] High-resolution mass spectrometry (ESI-MS) DAR value analysis of antibody-drug conjugate ADC-3

[0623] The molecular weight of ADC-3 was analyzed by a high-resolution mass spectrometer, and the deconvoluted mass spectrum is shown in Figure 16. Based on the measured molecular weight information, by comparing it with the theoretical molecular weight, the major molecular weight variants can be assigned, and the mass spectrum abundance of each major molecular weight variant is used to perform DAR value analysis, and the average DAR value is calculated to be 3.93.

[0624] Example 15: In vitro activity test of ADC-3 (BxPC-3, FaDu, HepG2)

[0625] Similar operations were performed using the method described in Reference Example 9 to test the inhibitory effect of the antibody-drug conjugate ADC-3 on cancer cell proliferation at various TROP2 expression levels at different concentration gradients. Human tumor cells, including BxPC-3, FaDu, and HepG2, were selected. The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 6 and Figures 17-19. ADC-3 and small molecule toxins both had significant inhibitory effects on TROP2-positive cells, while the TROP2 antibody, mAb-2, had no significant inhibitory effect on TROP2-positive cells. ADC-3 was significantly superior to monoclonal antibodies and small molecule toxins. Neither ADC-3 nor mAb-2 had an inhibitory effect on TROP2-negative cells, demonstrating good targeting.

[0626] Table 6. Inhibitory effects of different drugs on tumor cell proliferation (IC50, nM)

[0627] Example 16: In vivo activity testing of ADC-3 (NCI-N87CDX mouse model)

[0628] Referring to a method similar to that described in Example 10, the effect of ADC-3 on tumor growth inhibition in the NCI-N87CDX mouse model was evaluated. The tumor growth curves and body weight change curves after administration are shown in Figures 20-21. ADC-3 demonstrated good tumor growth inhibition and a good safety profile, and the experimental mice did not show any toxicity related to weight loss.

[0629] Example 17: Preparation and Characterization of ADC-4

[0630] Antibody-drug conjugate ADC-4 was prepared by referring to the above preparation method. The difference between ADC-4 and ADC-1 is that the antibody used is the anti-TROP2 antibody mAb-2. The characterization data of the antibody-drug conjugate ADC-4 are described below.

[0631] HIC-HPLC analysis of ADC-4, as shown in Figure 22, showed less than 5% unconjugated cytotoxic antibody; the conjugated product was primarily DAR2, with an overall DAR value of 1.89 for ADC-4. SEC-HPLC analysis of ADC-4 revealed less than 5% high-molecular-weight aggregates in the ADC drug substance, indicating that the ADC sample was primarily present in monomeric form.

[0632] Example 18: Preparation and Characterization of ADC-5

[0633] Antibody-drug conjugate ADC-5 was prepared by referring to the above preparation method. The difference between ADC-5 and ADC-1 is that the antibody is the anti-TROP2 antibody mAb-2, and the linker-payload is LP-2. The characterization data of ADC-5 are described below.

[0634] HIC-HPLC analysis of ADC-5, as shown in Figure 23, showed less than 5% unconjugated cytotoxic antibody; the conjugated product was primarily DAR2, with an overall DAR value of 1.87 for ADC-5. SEC-HPLC analysis of ADC-5 revealed less than 5% high-molecular-weight aggregates in the ADC drug substance, indicating that the ADC sample was primarily present in monomeric form.

[0635] Example 19: Comparison of in vitro activity of ADC-4 and ADC-5 (BxPC-3, FaDu, HepG2)

[0636] With reference to the method described in Example 9, the antibody-drug conjugates ADC-4 and ADC-5 were tested for their effects on cancer cell proliferation at various levels of TROP2 expression. TROP2-positive human tumor cells such as BxPC-3 (human pancreatic cancer cells) and FaDu (human laryngeal cancer cells), and TROP2-negative tumor cells such as HepG2 (human liver cancer cells) were selected. The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 7 and Figures 24-26. Among them, ADC-4, ADC-5, and the MMAE small molecule toxin all had a significant inhibitory effect on the proliferation of positive cells, and there was no significant difference in the activity of ADC-4 and ADC-5. The monoclonal antibody had no significant inhibitory effect on the proliferation of TROP2-positive cells. ADC-4, ADC-5, and the monoclonal antibody had no inhibitory effect on antigen-negative cells, reflecting good targeting.

[0637] Table 7. Inhibitory effects of different drugs on tumor cell proliferation (IC50, nM)

[0638] Example 20: Preparation and Characterization of ADC-6

[0639] The antibody-drug conjugate was prepared with reference to the above preparation method. The difference between it and ADC-1 is that the antibody used is mAb-3 (Trastuzumab) and the linker-payload is LP-6. The characterization data of the antibody-drug conjugate ADC-6 are described below. The HIC-HPLC analysis of ADC-6, the test results are shown in Figure 27, the unconjugated cytotoxic antibody is <5%; the conjugated product is mainly DAR4, and the overall DAR value of ADC-6 drug is 3.93. The SEC-HPLC test of ADC-6 shows that the high molecular weight polymer in the ADC drug is <5%, and the ADC sample mainly exists in the form of monomers.

[0640] Example 21: In vitro activity test of ADC-6 (SK-BR-3, NCI-N87)

[0641] Referring to the method described in Example 9, the antibody-drug conjugate ADC-6 was tested for its proliferative effects on various cancer cells expressing varying levels of HER2. Two HER2 cell lines, SK-BR-3 and NCI-N87, were selected to compare the activity of ADC-2 (containing the sortase recognition sequence) with that of ADC-6 (excluding the sortase recognition sequence). The results demonstrated no difference in cytotoxic activity between the target ADC and the absence of the SrtA-specific peptide recognition sequence (see Figures 28-29).

[0642] The sequences involved in this application are as follows:

[0643] SEQ ID No:1(Halo-Endo S2-His):

[0644] SEQ ID No: 2 (mAb-1 light chain):

[0645] SEQ ID No: 3 (mAb-1 heavy chain):

[0646] SEQ ID No: 4 (mAb-2 light chain):

[0647] SEQ ID No: 5 (mAb-2 heavy chain):

[0648] SEQ ID No: 6 (mAb-3 light chain):

[0649] SEQ ID No: 7 (mAb-3 heavy chain):

[0650] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A method for preparing a conjugated drug, characterized in that: The conjugated drug is site-specifically conjugated based on the N-glycosylation site of the Fc region, and the method comprises the following steps: (1) providing a donor containing an oxazoline oligosaccharide, a protein containing an Fc region, and an immobilized endoglycosidase having glycoside transfer activity, wherein the Fc region contains a GlcNAc motif; (2) covalently linking the donor containing oxazoline oligosaccharide to the protein containing Fc motif through the catalytic action of the endoglycosidase.

2. The method according to claim 1, characterized in that The protein containing the Fc region is an antibody or an Fc fusion protein.

3. A method for preparing an antibody-drug conjugate, characterized in that: The antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site in the Fc region of the antibody, and the method comprises the following steps: (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycoside transfer activity; (2) covalently linking the donor containing oxazoline oligosaccharide to the antibody containing the GlcNAc motif through the catalytic action of the endoglycosidase.

4. The method according to any one of claims 1 to 3, wherein the donor containing the oxazoline oligosaccharide further contains a load.

5. The method according to claim 4, wherein the loading substance is selected from the group consisting of small molecule compounds, agonists, nucleic acids, nucleic acid analogs, fluorescent molecules, radionuclides and immunomodulatory proteins; or The loading substance is selected from the group consisting of small molecule compounds (for example, small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapy drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine, etc.; small molecule targeted drugs, such as imatinib mesylate, gefitinib and anlotinib, etc.; immune agonists, such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radioactive labels, etc.), short peptides, polypeptides, peptidomimetics and proteins.

6. The method according to any one of claims 1 to 3, characterized in that The oxazoline oligosaccharide is selected from one or more of the following groups: disaccharide oxazoline, trisaccharide oxazoline, tetrasaccharide oxazoline, pentasaccharide oxazoline, hexasaccharide oxazoline, heptasaccharide oxazoline, octasaccharide oxazoline, nonasaccharide oxazoline, decasaccharide oxazoline and undecasaccharide oxazoline.

7. The method according to any one of claims 1 to 3, characterized in that The oxazoline oligosaccharide has the following structure: the first hexose group or its derivative-(the second hexose group or its derivative) f -β-D-pyranose glucoxazoline, f is 0, 1, 2, 3, 4, 5 or 6; the structure of β-D-pyranose glucoxazoline is:

8. The method according to claim 7, characterized in that The first hexosyl group or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or their derivatives; and / or The carbon at position 6 is in the form of -C(O)-; and / or The second hexosyl group or its derivative is independently selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives at each occurrence; and / or The monosaccharide moieties in the oligosaccharide structure are linked by β-(1→4) glycosidic bonds; and / or The first hexagonal glycosyl derivative and the second hexagonal glycosyl derivative are independently selected from uronic acid or a derivative in which the hydroxyl group of a monosaccharide is replaced by an acylamino group.

9. The method according to claim 7 or 8, characterized in that The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is a mannose group or a derivative thereof; or The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is a galactose group or a derivative thereof; or The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is a glucose group or a derivative thereof; or The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is a fructose group or a derivative thereof; or The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is a gulose group or a derivative thereof; or The oxazoline oligosaccharide has the following structure: a first hexose group or a derivative thereof-β-D-pyranose glucose oxazoline, wherein the first hexose group or a derivative thereof is an idose group or a derivative thereof.

10. The method according to claim 7 or 8, characterized in that The structure of the oxazoline oligosaccharide is:

11. A method for preparing a conjugated drug, characterized in that: The antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site in the Fc region, and the method comprises the following steps: (1) providing a donor containing an oxazoline oligosaccharide, a protein containing an Fc region, and an immobilized endoglycosidase having glycoside transfer activity, wherein the Fc region contains a GlcNAc motif; The donor containing oxazoline oligosaccharide is a linker-load compound of formula (I): (2) covalently linking the donor containing oxazoline oligosaccharides to the protein containing the Fc region through the catalytic action of the endoglycosidase; Wherein, the drug-drug conjugate structure is shown in formula (II): Wherein, P is the load; DC(O)-L- is a linker; The carbon 6 of the first hexose moiety or its derivative is in the form of -C(O)-, and f is 0, 1, 2, 3, 4, 5 or 6; L is a linker, and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1; R is hydrogen or α-L-fucosyl; q is 1 or 2; Protein is a protein containing an Fc region.

12. The method according to claim 11, characterized in that The first hexosyl group or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or their derivatives; and / or The second hexosyl group or its derivative is independently selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives at each occurrence; and / or The monosaccharide moieties in the oligosaccharide structure are linked by β-(1→4) glycosidic bonds; and / or The first hexagonal glycosyl derivative and the second hexagonal glycosyl derivative are independently selected from uronic acid or a derivative in which the hydroxyl group of a monosaccharide is replaced by an acylamino group.

13. The method according to claim 11, characterized in that The protein containing the Fc region is an antibody or an Fc fusion protein.

14. A method for preparing an antibody-drug conjugate, characterized in that: The antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site in the Fc region of the antibody, and the method comprises the following steps: (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycoside transfer activity; The donor containing oxazoline oligosaccharide is a linker-load compound of formula (I): (2) covalently linking the donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif through the catalytic action of the endoglycosidase; Wherein, the structure of the antibody-drug conjugate is as shown in formula (II-1), (II-2), (II-3), (II-4) or (II-5): Wherein, P is the load; DC(O)-L- is a linker; DC(O)- is a disaccharide structure; L is a linker, and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1; R is hydrogen or α-L-fucosyl; q is 1 or 2; Ab is an antibody or an antigen-binding fragment thereof.

15. A method for preparing an antibody-drug conjugate, characterized in that: The antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site in the Fc region of the antibody, and the method comprises the following steps: (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycoside transfer activity; The donor containing oxazoline oligosaccharide is a linker-load compound of formula (I): (2) covalently linking the donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif through the catalytic action of the endoglycosidase; The structure of the antibody-drug conjugate is shown in formula (II): Wherein, P is the load; DC(O)-L- is a linker; DC(O)- is a disaccharide structure L is a linker, and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1; R is hydrogen or α-L-fucosyl; q is 1 or 2; Ab is an antibody or an antigen-binding fragment thereof.

16. The method according to any one of claims 11 to 15, characterized in that Among them -L-(P) t -L 2 -L 1 -BP, that is, formula (I) is: in B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; L 1 independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination: 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent; 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or an integer from 10 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure; R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group; P is with B part, or L 1 Part, or L 2 Partially connected load.

17. The linker-load compound of any one of claims 11 to 15, wherein -L-(P)t is That is, formula (I) is: in, Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -CO-(PEG) on the side chain j -R 11 a substituted natural amino acid or an oligomeric natural amino acid having a degree of polymerization of 2-10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10); R 11 It is C 1-10 alkyl; d is 0, 1, 2, 3, 4, 5, or 6; -(PEG) i -and-(PEG) j - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 Alkylene; each i is independently an integer from 1 to 100, and each j is independently an integer from 1 to 100; M is hydrogen or LKa-L 2 ―L 1 ―B―P; Q is NH2 or L 2 ―L 1 ―B―P; The conditions do not include the following: M is hydrogen and Q is NH2; Each LKa is independently selected opSu is or a mixture thereof; wherein * represents and L 2 The connecting part; B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; L 1 independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination: 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent; 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-100 Integer, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid. m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure; R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group; P is with B part, or L 1 Part, or L 2 Partially connected load.

18. The method according to claim 16 or 17, characterized in that L 2 is the amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the disaccharide structure.

19. The method according to claim 18, characterized in that AA, at each occurrence, is independently any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His, or any combination thereof.

20. The method according to claim 16 or 17, characterized in that n is an integer of 1-100, preferably an integer of 1-50, preferably an integer of 1-30, preferably an integer of 1-20, preferably an integer of 1-10, and preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

21. The method according to claim 16 or 17, characterized in that L 1 The cleavable sequence comprises an amino acid sequence that can be cleaved by an enzyme, wherein the amino acid sequence that can be cleaved by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

22. The method according to claim 21, characterized in that Among them L 1 It is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably, it is -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof.

23. The method according to claim 16 or 17, characterized in that L 1 For -Val-Cit-.

24. The method according to claim 16 or 17, characterized in that Wherein B is selected from: (-PABC-), -NH-CH2-U-, or -NH-CH2-U-(CH2) g -(CO)-; wherein g is 1, 2, 3, 4, 5 or 6; U is absent or is CH2, O, S or NH, preferably O or S.

25. The method according to claim 16 or 17, characterized in that Where -L 1 -B- stands for -Val-Cit-PABC-.

26. The method according to claim 16 or 17, characterized in that Where -L 2 -L 1 -B- represents -Gly-Gly-Gly-Val-Cit-PABC- or -HN-(C2H4-O)m-(CH2)p-Gly-Gly-Phe-Gly-.

27. The method according to any one of claims 11 to 15, characterized in that The load P is selected from the group consisting of small molecule compounds (for example, small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapy drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine, etc.; small molecule targeted drugs, such as imatinib mesylate, gefitinib and anlotinib, etc.; immune agonists, such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radioactive labels, etc.), short peptides, polypeptides, peptidomimetics and proteins.

28. The method according to any one of claims 11 to 15, characterized in that The cargo P is a cytotoxin or a fragment thereof, optionally derivatized to attach to the L moiety in the compound of formula (I); Preferably, the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhy-drovinblastine, dolastatin 10, 10) and its analogs, halichondrin B, eribulin, indole-3-oxyacetamides, podophyllotoxins, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide, camptothecins and their derivatives, mitoxantrone, mitoguanidine, nitrogen mustards, nitrosoureas, aziridines, benzodopa, carboquinone, metodepa, uredepa, dynemicin, esperamicin (esperamicin), neocarzinostatin, aclarubicin, dactinomycin, anthramycin, bleomycin, actinomycin C, caramidocin, carminomycin, carminomycin, dactinomycin D, daunorubicin, detoxib, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycins, noramycin, olivomycin, peplomycin, porfiromycin, puromycin, ferroxorubicin, rhodorubicin, streptozocin, streptozocin, zorubicin, trichothecenes, T-2 toxin, verracurin A, baculosporin A, anguidine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, dimethylfolate, methotrexate, pteropterin, trimetrexate, edatrexate, fludarabine, 6-mercaptopurine, thiamidine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, dooxyfluridine, floxuridine, captestosterone, drostanolone propionate, cyclothiosteroid, methotrexate, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprolide acetate, protein kinase inhibitors and proteasome inhibitors; and / or selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine, and exatecan, and derivatives thereof; and / or selected from auristatins, in particular MMAE, MMAF or MMAD; and / or Selected from exatecan and its derivatives, such as DX8951f; and / or Selected from DXd-(1) and DXd-(2); preferably DXd-(1).

29. The method according to any one of claims 11 to 14, characterized in that The linker-load compound is:

30. The method according to any one of claims 1 to 29, characterized in that The protein containing the Fc region in the drug conjugate is an antibody, and the antibody in the drug conjugate or the antibody drug conjugate is an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD25 antibody, an anti-CD30 / TNFRSF8 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD44v6 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-CD74 antibody, an anti-CD79b antibody, an anti-CD117 / KITk antibody, an anti-CD123 antibody, an anti-CD138 antibody, an anti-CD142 antibody, an anti-CD174 antibody, an anti-CD227 / MUC1 antibody, an anti-CD352 antibody, an anti-CLDN18.2 Antibodies, anti-DLL3 antibodies, anti-ErbB2 / HER2 antibodies, anti-CN33 antibodies, anti-GPNMB antibodies, anti-ENPP3 antibodies, anti-Nectin-4 antibodies, anti-EGFRvⅢ antibodies, anti-SLC44A4 / AGS-5 antibodies, anti-CEACAM5 antibodies, anti-PSMA antibodies, anti-TIM1 antibodies, anti-LY6E antibodies, anti-LIV1 antibodies, anti-Nectin4 antibodies, anti-SLITRK6 antibodies, anti-HGFR / cMet antibodies, anti-SLAMF7 / CS1 antibodies, anti-EGFR antibodies, anti-BCMA antibodies, anti-AXL antibodies, anti-NaPi2B antibodies, anti-GCC antibodies, anti-STEAP1 antibodies, anti-MUC16 antibodies, anti-mesothelin antibodies, anti-ETBR antibodies, anti-EphA2 antibodies, anti-5T4 antibodies, anti-FOLR1 antibodies, anti-LAMP1 antibodies, anti-Cadherin 6 antibodies, anti-FGFR2 antibodies, anti-FGFR3 antibodies, anti-CA6 antibodies, anti-CanAg antibodies, anti-integrin αV antibodies, anti-TDGF1 antibodies, anti-ephrin A4 antibodies, anti-TROP2 antibodies, anti-PTK7 antibodies, anti-NOTCH3 antibodies, anti-C4.4A antibodies, anti-FLT3 antibodies, anti-B7H3 / 4 antibodies, anti-TF (tissue factor) antibodies, anti-ROR1 / 2 / antibodies; preferably anti-CD19 antibodies, anti-ErbB2 / HER2 antibodies, anti-CLDN18.2 antibodies, anti-Nectin-4 antibodies, anti-FGFR3 antibodies, anti-Trop2 antibodies.

31. The method according to any one of claims 1 to 30, characterized in that The endoglycosidase with glycoside transfer activity is N-acetylglucosamine endohydrolase.

32. The method according to claim 31, characterized in that The N-acetylglucosamine endohydrolase comprises at least one selected from the group consisting of Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd) and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); or The N-acetylglucosamine endohydrolase includes at least one selected from Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S and Endo S2.

33. The method according to any one of claims 1 to 30, characterized in that The endoglycosidase with glycoside transfer activity is covalently linked to a Halo tag and is immobilized on a support containing a haloalkyl linker through the Halo tag. The Halo tag is a dehalogenase or a variant thereof or a truncated functionally active portion thereof.

34. The method according to claim 33, wherein One end of the endoglycosidase is covalently linked to a Halo tag, and the other end is covalently linked to a His tag; or The amino terminus of the endoglycosidase is covalently linked to a Halo tag, and the carboxyl terminus is covalently linked to a His tag, i.e., Halo-endoglycosidase-His; or The amino terminus of the endoglycosidase is covalently connected to a Halo tag, and the carboxyl terminus is covalently connected to a His tag. The endoglycosidase is Endo-S2, namely Halo-Endo S2-His.

35. The method according to claim 33, wherein The support comprises a chloroalkyl linker, such that the endoglycosidase is immobilized on the support via a covalent interaction between the chloroalkyl linker and the Halo tag.

36. The method according to claim 35, characterized in that The chloroalkyl linker is generated from a chloroalkyl substrate having the structure of formula (III): Wherein, u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19.

37. The method according to claim 33, wherein The support has the structure of formula (IV): Wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19; It is a resin, bead, membrane, gel, matrix, film, plate, well, tube, slide or surface, preferably a resin, more preferably agarose resin, silicone resin, polymethyl methacrylate resin or cellulose resin, most preferably highly cross-linked agarose resin or polymethyl methacrylate.

38. A glycosidase fusion protein, characterized in that The invention comprises a covalently linked endoglycosidase and a Halo tag, wherein the Halo tag is a dehalogenase or a variant thereof or a truncated functionally active portion thereof.

39. The endoglycosidase fusion protein according to claim 38, characterized in that The amino terminus of the endoglycosidase is covalently connected to a Halo tag, and the carboxyl terminus of the endoglycosidase is covalently connected to a His tag.

40. The endoglycosidase fusion protein according to claim 38 or 39, characterized in that The endoglycosidase is selected from at least one of Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd) and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); or The endoglycosidase is selected from at least one of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S and Endo S2.

41. The endoglycosidase fusion protein according to claim 38 or 39, characterized in that The endoglycosidase fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, or has at least 80% identity, at least 90% identity with SEQ ID NO: 1, or has one or more conservative amino acid substitutions with SEQ ID NO:

1.

42. The endoglycosidase fusion protein according to claim 38 or 39, characterized in that The PI of the endoglycosidase fusion protein is about 4-7.

43. An immobilized endoglycosidase fusion protein, characterized in that A method comprising immobilizing the endoglycosidase fusion protein of any one of claims 38 to 42 on a support.

44. The immobilized endoglycosidase fusion protein according to claim 43, characterized in that The support comprises a haloalkyl linker, so that the endoglycosidase fusion protein is immobilized on the support through the covalent interaction between the haloalkyl linker and the Halo tag. Preferably, the haloalkyl linker is a chloroalkyl linker.

45. The immobilized endoglycosidase fusion protein according to claim 44, characterized in that The chloroalkyl linker is generated from a chloroalkyl substrate having the structure of formula (III): Wherein, u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19.

46. ​​The immobilized endoglycosidase fusion protein according to claim 44, characterized in that The support has the structure of formula (IV): Wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19; It is a resin, bead, membrane, gel, matrix, film, plate, well, tube, slide or surface, preferably a resin, more preferably agarose resin, silicone resin, polymethyl methacrylate resin or cellulose resin, most preferably highly cross-linked agarose resin or polymethyl methacrylate.

47. A prepacked column, characterized in that The prepacked column is filled with the immobilized endoglycosidase fusion protein according to any one of claims 43-46.

48. Use of the immobilized endoglycosidase fusion protein according to any one of claims 43 to 46 and / or the prepacked column according to claim 47 in the preparation and / or purification of a conjugated drug or antibody conjugate.

49. Use of a coupling device in implementing the method according to any one of claims 1 to 37, the coupling device comprising a flow reactor and a fluid transport unit, the flow reactor being filled with an immobilized endoglycosidase, the fluid transport unit being connected to the inlet fluid of the flow reactor and transporting a donor containing an oxazoline oligosaccharide and an antibody containing a GlcNAc motif to the flow reactor.

50. The use according to claim 44, characterized in that The coupling device comprises: at least one flow reactor having an inlet and an outlet, wherein the flow reactor is filled with a medium and the endoglycosidase is immobilized on the medium; a fluid delivery unit in fluid communication with an inlet of the flow reactor and configured to continuously supply at least one reaction fluid to the flow reactor according to different stages of the coupling process, the at least one reaction fluid comprising a donor containing an oxazoline oligosaccharide and an antibody containing a GlcNAc motif or a protein containing an Fc region; and The fluid collecting unit is in fluid communication with the outlet of the flow reactor and is configured to control the collection of the fluid flowing out of the outlet of the flow reactor according to different stages of the coupling process.

51. The use according to claim 49 or 50, characterized in that The at least one reaction fluid comprises a first reaction fluid and a second reaction fluid, the buffer, the first reaction fluid and the second reaction fluid are stored in a first container, a second container and a third container, respectively, the fluid delivery unit comprises a first delivery pump and a second delivery pump, wherein the first container and the second container are connected to the first delivery pump via a first container outlet pipe and a second container outlet pipe, respectively, the third container is connected to the second delivery pump via a third container outlet pipe, the first delivery pump and the second delivery pump are connected to an inlet main pipe via a first inlet branch pipe and a second inlet branch pipe, respectively, and the inlet main pipe is connected to the inlet of the flow reactor, Furthermore, during pre-reaction equilibration, post-reaction recovery, and post-recovery flushing, the first delivery pump pumps the buffer solution in the first container into the inlet main pipe; during the coupling reaction, the first delivery pump pumps the first reaction fluid in the second container into the inlet main pipe, and the second delivery pump pumps the second reaction fluid in the third container into the inlet main pipe.

52. The use according to any one of claims 49 to 51, characterized in that The coupling device also includes: a sampling and detection unit, which is connected to the outlet fluid of the flow reactor and is configured to: collect sample fluid from the fluid flowing out of the outlet of the flow reactor according to a preset sampling time; and detect the conjugate in the sample fluid to obtain a detection result, which indicates whether the conjugate meets the preset standard.

53. The use according to claim 52, characterized in that The coupling device further includes: a recycling unit, which is arranged between the inlet and the outlet of the flow reactor; when the detection result indicates that the conjugate does not meet the preset standard, the fluid collection unit is configured to stop collecting the fluid flowing out of the outlet of the flow reactor, and the recycling unit is configured to control the fluid flowing out of the outlet of the flow reactor to re-enter the inlet of the flow reactor to perform the coupling reaction again in the flow reactor; and / or The sampling and detection unit further includes a sampling pump, a first switching valve, an elution pump, at least one analytical column, and a detector. The sampling pump is connected to the outlet of the flow reactor via a sampling line. The first switching valve is provided with a sample loop, and the first switching valve switches between a first state and a second state according to a preset sampling time. When the first switching valve is in the first state, the sampling pump is in fluid communication with the sample loop, collects sample fluid from the fluid flowing out of the outlet of the flow reactor via the sampling line, and pumps the sample fluid into the sample loop. When the first switching valve is in the second state, the elution pump, the sample loop, the at least one analytical column, and the detector are in fluid communication via the detection line. The elution pump pumps eluent into the detection line, causing the eluent to flow through the sample loop, thereby driving the sample fluid in the sample loop to flow through one of the at least one analytical column and then into the detector.

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